Methods for aptamer selection

ABSTRACT

The present disclosure relates to methods for identifying aptamers against allergen proteins and signaling polynucleotides (SPNs) for allergen detection. The screening method of the present disclosure combines several positive SELEX selections, and on-chip positive and counter selections to identify aptamer sequences that are preferentially bind to target proteins when competing with short complementary sequences.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application No. 62/714,102 filed Aug. 3, 2018, entitled with “Methods for Aptamer Selection”; the contents of which are incorporated herein by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The instant application is being filed along with a Sequence Listing text file in electronic format. The Sequence Listing is provided as a file entitled 2066_1011PCT_SL.txt, created on Aug. 1, 2019, which is 14,790,207 bytes in size. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to methods for identification of aptamers against a target of interest (e.g., an allergen). The disclosure also provides aptamers, signaling polynucleotides (SPNs), DNA chips, detection sensors and kits, and assays for detecting a target in a sample.

BACKGROUND OF THE DISCLOSURE

Nucleic acid aptamers are single-stranded oligonucleotides (DNAs, RNAs or DNA/RNA hybrids) that can bind to target molecules with high affinity and specificity. Nucleic acid aptamers are generally selected from a library of oligonucleotides with randomized sequences by an iterative process of adsorption, recovery and reamplification, for example, by conventional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and other closely related methods (See, e.g., U.S. Pat. Nos. 5,270,163; 5,567,588; 5,637,459; 5,670,637; 5,705,337; and 5,723,592). Aptamers can adapt unique secondary and tertiary structures and recognize targets with high affinity and specificity.

Aptamers provide a cost-effective alternative to antibodies as there is no need for aptamer selection in animals or cell lines, they have shelf-lives of years, and they can be easily modified to reduce cross-reactivity with undesired molecules. Aptamers have significant advantages over antibodies, such as better specificity and affinity, wider varieties of targets, easier synthesis and modification, higher stability and lower cost. These properties favor aptamers as new detection agents for wide applications in biosensor development, among other fields, for detecting the presence, absence and/or amount of target molecules in a sample. For example, aptamers and aptamer-based assays have been shown, among many other useful applications (e.g., diagnostic tests and therapy) as a promising alternative in food safety control, e.g., detection and control of pathogens, toxins, allergens and other forbidden contaminants in food matrices (Amaya-Gonzalez, et al., Sensors, 2013, 13:16292-16311; and Amaya-Gonzalez, et al., Anal. Chem. 2014, 86(5), 2733-2739). Aptamers based assays replace many immunoblotting methods using antibodies (e.g., ELISA).

Allergy (e.g., food allergy) is a common medical condition. It has been estimated that in the United States, up to 2 percent of adults and up to 8 percent of children, particularly those under three years of age, suffer from food allergies (about 15 million people), and this prevalence is believed to be increasing. Allergen detection, either in clinical settings or consumer based, is important to a person who is allergic to certain types of food, e.g., gluten and peanuts. Sensitive and specific detection agents against allergens are keys in developing detection assays that can efficiently and quickly test a suspect food product before consuming it. Aptamers that selectively bind to an allergen have been employed in many allergen detection sensors and assays (Weng and Neethirajan, Biosens Bioelectron, 2016, 85: 649-656; Svobodova et al., Food Chem., 2014, 165: 419-423; Tran et al., Biosens. Bioelectron, 2013, 43, 245-251; and Nadal et al., Plos One, 2012, 7(4): e35253). Studies have shown that an aptamer-based assay has significant advantages as compared to antibody-based immunoassay (e.g., ELISA).

The present disclosure developed a modified selection method for identifying aptamer sequences against a specific allergen target; the aptamers and/or signal polynucleotides derived from the aptamers can be directly used in detection assays with increased specificity and sensitivity. Specifically, the modified selection method combines several positive, negative and counter selection processes to identify aptamers that can specifically recognize a target molecule (e.g., an allergen protein) but the features (e.g., the primary and secondary structures) of the aptamers block the same aptamers bound to the target to hybridize to short oligonucleotides comprising sequences complementary to the same aptamers. Therefore, the target and the short complementary sequence do not bind to the same aptamer simultaneously.

SUMMARY OF THE DISCLOSURE

The present disclosure provides screening methods tailored for selection of aptamers against target molecules that can be directly employed in competition-based target detection assays, such as allergen detection assays; the methods comprising several positive, negative (counter) selection processes to identify aptamers having particular primary and secondary structural features that when the aptamers are bound to target molecules to form aptamer.target complexes, they do not simultaneously hybridize to short oligonucleotides complementary to the aptamer sequences. The identified aptamers are suitable to develop chip sensors for target detection in which the aptamers or signal polynucleotides derived from the aptamers compete binding to their target molecule in the presence of oligonucleotides (i.e., anchor sequences) that are complementary to the sequences of the aptamers.

In some embodiments, the screening method comprises (a) preparing an input DNA library comprising a plurality of single stranded DNA (ssDNA) molecules, each of which comprises a central randomized nucleic acid sequence flanked by a constant sequence at the 5′ end and a constant sequence at the 3′ end, the constant 5′ end and the constant 3′ end functioning as primers; (b) selecting a pool of ssDNA molecules, from the input DNA library of (a), that substantially bind to a target material; (c) selecting a pool of ssDNA molecules, from the target binding pool of ssDNA molecules obtained in (b), that do not bind to the complementary sequences in the presence of the target material (i.e., do not simultaneously bind to the target and complementary sequences); (d) counter-selecting ssDNA molecules, from the positive binding pool of ssDNA molecules obtained in (c), that do not bind to the complementary sequences in the absence of the target material, or that substantially bind to counter target materials; and (e) subtracting the pool of ssDNA molecules obtained in (d) from the positive binding pool of ssDNA molecules in (c), and identifying candidate ssDNA molecules that specifically bind to the target of interest. Each sub-pool of ssDNA molecules can be identified through positive SELEX and/or on-chip selection processes and each process can be repeated several rounds at the same condition.

Accordingly, the aptamers identified via the present screening methods and signaling polynucleotides (SPNs) derived from the aptamers bind to their target molecule with high affinity and specificity. In some embodiments, the aptamers and SPNs may not hybridize to short complementary sequences in the presence of the target molecule, while they can bind to the short complementary sequences in the absence of the target molecule.

In some embodiments, the present screening method further comprises amplifying the ssDNA molecules in each pool after each selection process. The ssDNA molecules may be amplified by PCR using a pair of primers labeled with a fluorophore probe. The amplified and regenerated ssDNA molecules are therefore labeled with the fluorophore probe.

In some embodiments, the pool of ssDNA molecules that substantially bind to a target may be selected by a modified Graphene Oxide (GO)-SELEX process using an input ssDNA library and a target material. This positive selection may comprise the steps of (i) contacting the input ssDNA library with the target material wherein complexes are formed between the target and a plurality of ssDNA molecules present in the input library; (ii) partitioning the complexes formed in step (i) using a Graphene Oxide (GO) solution, and isolating the ssDNA molecules in the complexes to produce a subset of ssDNA molecules for the target material; (iii) contacting the subset of ssDNA molecules in (ii) with the same target material wherein complexes are formed between the target and a second plurality of ssDNA molecules present in the subset of ssDNA molecules to generate a second subset group of ssDNA molecules; and (iv) optionally repeating steps (ii) to (iii), one, two, three or more times to produce a respective third, fourth, fifth or more subset group of ssDNA molecules, thereby producing the enriched pool of ssDNA molecules that substantially bind to the target material.

In some embodiments, the positive pool of ssDNA molecules that do not bind to the complementary sequences in the presence of the target material may be selected through on-chip positive binding selection process using the target binding pool of ssDNA molecules (e.g., the pool of ssDNA molecules selected by the GO-SELEX process), the same target material and a solid support that is coated with a plurality of short oligonucleotides comprising sequences complementary to the sequences of the ssDNA molecules, e.g., the constant sequence at the 5′ end of the ssDNA molecules.

In some embodiments, the positive binding pool of ssDNA molecules selected by the on-chip positive selection process may be further refined to subtract non-specific ssDNA molecules. The counter selection may comprise: (i) counter selecting a pool of ssDNA molecules, from the positive pool of ssDNA molecules (e.g., the pool from the on-chip positive selection), that do not bind to the complementary sequences even in the absence of the target material (i.e. the non-binding ssDNA molecules); this selection including an on-chip non-binding counter process that uses the positive binding pool of ssDNA molecules as the input and a chip that is coated with short oligonucleotides comprising complementary sequences of the ssDNA molecules; and (ii) counter selecting a pool of ssDNA molecules, from the positive binding pool of ssDNA molecules, that substantially bind to counter target molecules; this selection including an on-chip counter binding process using the positive binding pool of ssDNA molecules as the input, one or more counter target materials and a chip that is coated with short oligonucleotides comprising sequences complementary to the sequences of the ssDNA molecules.

In some embodiments, the target material may be a common allergen such as a common food allergen. In one embodiment, the target material is peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio, walnut, gluten, whey and/or casein.

In another aspect, the present disclosure provides aptamers, signaling polynucleotides (SPNs), DNA chips, aptamer-based detection sensors and kits for detecting the presence, absence, and/or amount of a target (e.g., an allergen) in a sample.

In some embodiments, aptamer sequences that specifically bind to an allergen are selected by the present selection processes, wherein the allergen is a common food allergen, e.g., peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio, walnut, gluten, whey and casein. Aptamers that can bind to all nuts including peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut, may also be selected, for example, by multiple SELEX methods.

In some embodiments, aptamer sequences that specifically bind to peanut are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs.3 to 1002. In some examples, the aptamer against peanut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1003 to 4002.

In some embodiments, aptamer sequences that specifically bind to almond are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID Nos. 4003 to 5002. In some examples, the aptamer against almond may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5003 to 8002.

In some embodiments, aptamer sequences that specifically bind to brazil nut are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 8003 to 9002. In some examples, the aptamer against brazil nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9003 to 12002.

In some embodiments, aptamer sequences that specifically bind to cashew are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 12003 to 13002. In some examples, the aptamer against cashew may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 13003 to 16002.

In some embodiments, aptamer sequences that specifically bind to hazelnut are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 16003 to 17002. In some examples, the aptamer against hazelnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 17003 to 20002.

In some embodiments, aptamer sequences that specifically bind to pecan are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 20003 to 21002. In some examples, the aptamer against pecan may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 21003 to 24002.

In some embodiments, aptamer sequences that specifically bind to pistachio are selected which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 24003 to 25002. In some examples, the aptamer against pistachio may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 25003 to 28002.

In some embodiments, aptamer sequences that specifically bind to walnut are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28003 to 29002. In some examples, the aptamer against walnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 29003 to 32002.

In some embodiments, aptamer sequences that can bind to all nuts are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 32003 to 33002. In some examples, the aptamer against all nuts may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 33003 to 36002.

In some embodiments, aptamer sequences that specifically bind to gluten are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 40003 to 41002. In some examples, the aptamer against gluten may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 41003 to 44002.

In some embodiments, aptamer sequences that specifically bind to whey are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 44003 to 45002. In some examples, the aptamer against whey may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 45003 to 48002.

In some embodiments, aptamer sequences that specifically bind to casein are selected, which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 48003 to 49002. In some examples, the aptamer against casein may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 49003 to 52002.

In some embodiments, aptamer sequences that specifically bind to a target control material may be selected. Such control sequences can be used together with the aptamer sequences that bind to the target in a detection assay. The control aptamer sequences have similar response to the sample (e.g., the food matrix) as the target specific aptamers. However, the control aptamers will not respond to the target (e.g., a target allergen) and have no binding affinity to the target specific aptamers or to the short anchor sequences complementary to the target specific aptamers. For example, aptamer sequences that bind to peanut control material may be used together with the aptamer sequences against peanut for detecting the presence/absence of peanut in a food sample. The peanut control sequences and the aptamer specific to peanut may demonstrate a similar response to the food type to be tested. Therefore, the signal from the peanut control sequences can be used as internal sample control.

In some examples, aptamer sequences that bind to peanut control material are selected which may comprise a unique nucleic acid sequence selected from the group consisting of SEQ ID NOs. 36003 to 37002. In some examples, the aptamer sequence for peanut control may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 37003 to 40002.

In accordance with the present disclosure, a SPN may comprise an aptamer selected by the present method that specifically binds to a target of interest and a short nucleic acid sequence that is complementary to the aptamer sequence. The short complementary sequences may be printed on a solid surface for a detection assay. In some embodiments, the short complementary sequence may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 52003 to 52042.

In further another aspect, the present disclosure provides methods for detecting the presence, absence and/or amount of a target in a sample using aptamers and SPNs identified by the present screening methods. In some embodiments, the target is a food allergen and the sample to be tested is a food sample. The food allergen may be peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio, walnut, gluten, whey and casein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart demonstrating an embodiment of the aptamer screening methods of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control.

The present screening methods modify conventional aptamer selection methods, combining several positive and negative (counter) selections to identify aptamers that specifically bind to a target of interest. These selections mimic the conditions of competition-based detection assays in which aptamers (or SPNs derived from the aptamers) are used to capture their target and short oligonucleotides comprising sequences complementary to the aptamers are used to detect the presence or absence of the aptamer:target complexes. The competition particularly is between the target to which an aptamer can bind with high level of specificity and affinity, and complementary sequences of the aptamer. The selected aptamer sequences can specifically bind to their target, but only hybridize to short complementary sequences in the absence of the target. The selected aptamer sequences cannot bind to the short complementary sequences in the presence of their target. The present screening methods also select control aptamer sequences for a specific target material. The control aptamer sequences can be used in parallel with target specific aptamers and serve as internal control. The detailed description of the screening methods is included.

Definitions

In order for the present disclosure to be more readily understood, certain terms and phrases are defined below. Additional terms and phrases are also defined and set forth through the specification.

As used herein, the term “aptamer” refers to a nucleic acid molecule or a peptide that can bind to a specific target molecule. A nucleic acid aptamer is a nucleic acid molecule having at least one binding site for a target molecule, such as another nucleic acid sequence, protein, peptide, antibody, small organic molecule, mineral, cell and tissue. A nucleic acid aptamer can be a single stranded or double stranded deoxyribonucleic acid (ssDNA or dsDNA), or ribonucleic acid (RNA), or a hybrid of DNA/RNA. Nucleic acid aptamers typically range from 10-150 nucleotides in length, for example, from 15-120 nucleotides in length, or from 20-100 nucleotides in length, or from 20-80 nucleotides in length, or from 30-90 nucleotides in length, or from 50-90 nucleotides in length. The nucleic acid sequence of an aptamer may optionally have a minimum length of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. In the context of the present disclosure, the term “aptamer” refers to a nucleic acid aptamer. The terms “a single stranded DNA(ssDNA) molecule,” and “aptamer” are used interchangeably.

An aptamer can fold into specific and stable secondary, tertiary, or quaternary conformational structures that enable it to bind to a target with high specificity and affinity. The structures may include, but are not limited to, hairpin loop, bulge loop, internal loop, multi-branch loop, pseudoknot, or combinations thereof. For example, the binding site of an aptamer may comprise a stem loop conformation or G-quartets.

Aptamers against a target may be naturally occurring or made by synthetic or recombinant means. An aptamer can be selected from a random oligonucleotide library through repeated rounds of in vitro partition, selection and amplification of nucleic acid molecules, e.g., conventional SELEX. As used herein, the term “SELEX” refers to a methodology known in the art as “Systematic Evolution of Ligands by Exponential Enrichment (SELEX)”. SELEX, or equivalently In vitro selection, is a powerful and widely used method to select nucleic acid sequences (i.e., aptamers) that bind to a target (e.g., a protein) with specificity and affinity (Ellington A D, et al., Nature, 1990, 346: 818-822; Tuerk C, et al., Science, 1990, 249: 505-510; and Gold L, et al., Anmi Rev Biochem, 1995, 64: 763-797). The SELEX process and various modifications are described in the art, e.g., U.S. Pat. Nos. 5,270,163; 5,567,588; 5,696,249; 5,853,984; 6,083,696; 6,376190; 6, 262, 774; 6,569,620; 6,706,482; 6,730,482; 6,933,116; 8,975,388; 8,975026; and 9,382,533; the contents of each of which are incorporated herein by reference in their entirety. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (i.e., form specific binding complexes) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. SELEX relies as a starting point upon a large library of single stranded oligonucleotides comprising randomized sequences. The oligonucleotides can be modified or unmodified DNAs, RNAs, or DNA/RNA hybrids. In some examples, the library comprises 100% randomized or partially randomized oligonucleotides.

Nucleic acid aptamers show robust binding affinities to their target, preferably binding to the target with an equilibrium (K_(d)) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamers also bind to the target molecule with a very high degree of specificity. It is preferred that aptamers have a K_(d) with the target molecule at least 10, 100, 1000, 10,000, or 100,000-fold lower than the K_(d) of other non-targeted molecules. In some examples, the aptamer selection process may be tailored to select aptamers with pre-defined parameters such as equilibrium (K_(d)), rate constants (K_(off) and K_(on)) and thermodynamic parameters (ΔH and ΔS) of aptamer-target interaction.

Aptamers may comprise naturally occurring nucleotides, and/or modified nucleotides including but not limited to chemically modified nucleobases, unnatural bases (e.g., 2-aminopurine), nucleotide analogs, addition of a label (e.g., a fluorophore), addition of a conjugate, or mixtures of any of the above. The nucleic acid sequence of an aptamer can be modified as desired so long as the functional aspects are still maintained (e.g., binding to the target).

As used herein, the terms “nucleic acid”, “oligonucleotide” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides of any length, and such nucleotides may include deoxyribonucleotides (DNAs), ribonucleotides (RNAs), and/or analogs or chemically modified deoxyribonucleotides or ribonucleotides and RNA/DNA hybrids. The terms “nucleic acid”, “oligonucleotide” and “polynucleotide” include double- or single-stranded molecules as well as triple-helical molecules. A nucleic acid molecule may comprise at least one chemical modification.

As used herein, the term “primary structure” of a nucleic acid molecule refers to its nucleotide sequence. The “secondary structure” of a nucleic acid molecule include, but is not limited to, a hairpin loop, a bulge loop, an internal loop, a multi-branch loop, a pseudoknot or combinations thereof. “Pre-selected secondary structures” refer to those secondary structures that are selected and engineered into an aptamer by design.

As used herein, the term “complementary” refer to the natural binding of polynucleotides by base pairing such as A-T(U) and C-G pairs. Two single-stranded molecules may be partially complementary such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. As used herein, the term “hybridization” or “hybridize to” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under permissive annealing conditions.

As used herein, the term “high affinity” refers to the binding of a candidate aptamer to a target with binding dissociation constant K_(a) less than 100 nM. The “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other components in a test sample. In similar, the term “specifically binds” means that an aptamer reacts or associates more frequently, more rapidly, with greater duration and with greater affinity with a particular target molecule, than it does with non-target molecules. For example, an aptamer against a target allergen binds to that allergen or a structural part or fragment thereof with greater affinity, avidity, more readily, and/or with greater duration than it binds to unrelated allergen proteins and/or parts or fragments thereof. It is also understood by reading this definition that, for example, an aptamer that specifically binds to a first target may or may not specifically bind to a second target. As such, “specific binding” does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term “selective binding”. The specificity of binding is defined in terms of the comparative dissociation constants (K_(d)) of the aptamer for its target as compared to the dissociation constant with respect to the aptamer and other materials in the environment or unrelated molecules in general. Typically, the K_(d) for the aptamer with respect to the target will be 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold less than the K_(a) with respect to the target and the unrelated molecule or accompanying molecule in the environment. Even more preferably, the K_(a) will be 50-fold, 100-fold, 150-fold or 200-fold less.

As used herein, the term “amplification” or “amplifying” means any process or combination of steps that increases the amount or number of copies of a molecule or class of molecules. The amplification of a nucleic acid molecule is generally carried out but not limiting to using polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; the contents of each of which are herein incorporated by reference in their entirety).

As used herein, the term “library,” or “pool,” or “subset” refers to a plurality of compounds, e.g. single stranded DNA (ssDNA) molecules.

As used herein, the terms “target molecule,” “target material” and “target” are used interchangeably to refer to any molecule to which an aptamer can bind. “Target molecules” or “targets” can be, for example, proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, small molecules, dyes, nutrients, pollutants, growth factors, cells, tissues, or microorganisms and any fragment or portion of any of the foregoing. In one embodiment, a target may be an allergenic protein.

As used herein, the term “counter target” refers to a molecule belonging to a family which has a similar structure, a similar active site, or similar activity to a target or a target material. In the context of the present disclosure, a counter target can be any molecules to which a selected aptamer against a target of interest has no cross-specificity. Counter targets may be used in counter selection processes to refine aptamer candidates for separating sequences that cross-recognize other closely related molecules.

As used herein, the term “allergen” means a compound, substance or composition that causes, elicits or triggers an immune reaction in a subject. As such, allergens are typically referred to as antigens. An allergen is typically a protein or a polypeptide.

As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.

As used herein, the term “sample” means a composition that contains or is assumed to contain one or more targets to be tested. A sample may be, but is not limited to, a biological sample obtained from a subject (including human and animal), a sample obtained from the environment (e.g., soil sample, water sample, agriculture sample such as a plant and a crop sample), a chemical sample, and a food sample.

Combined Selection Processes

In accordance with the present disclosure, the selection method is modified to identify aptamer candidates that can recognize a target molecule with high specificity and affinity and lower cross-reactivity with counter targets, and that do not hybridize to oligonucleotides that are complementary to the aptamer sequences in the presence of the target. The selected aptamers and SPNs derived from these aptamers can be used as detection agents in competition-based detection assays in which target molecules in a test sample and the complementary oligonucleotides compete binding to the aptamers (or SPNs).

In accordance with the present disclosure, the aptamer screening method may comprise (a) preparing an input DNA library comprising a plurality of single stranded DNA (ssDNA) molecules, each of which comprises a central randomized nucleic acid sequence flanked by a constant sequence at the 5′ end and a constant sequence at the 3′ end, the constant 5′ end and the constant 3′ end functioning as primers; (b) selecting a pool of ssDNA molecules, from the input DNA library of (a), that substantially bind to a target material; (c) selecting a pool of ssDNA molecules, from the target binding pool of ssDNA molecules obtained in (b), that do not bind to the complementary sequences in the presence of the target material (i.e., do not simultaneously bind to the target and complementary sequences); (d) counter-selecting ssDNA molecules, from the positive binding pool of ssDNA molecules obtained in (c), that do not bind to the complementary sequences in the absence of the target material (referred to as non-binding ssDNA molecules), or that substantially bind to counter target materials (cross-specificity); and (e) subtracting the pool of ssDNA molecules obtained in (d) from the positive binding pool of ssDNA molecules in (c), and identifying candidate ssDNA molecules that specifically bind to the target of interest.

The present screening methods combine several positive target binding selections (e.g., positive SELEX and on-ship SELEX), non-binding counter selections, complementary hybridization selections and counter target binding selections. Candidate aptamers are identified through repeated positive and negative selections, sequence amplification and sequencing analysis. The modified screening method affords improved efficiency in aptamer selection as compared to conventional SELEX and other known methods in the art and ensures selection of aptamers that preferably bind to a target molecule to short complementary nucleic acid sequences. The flow chart in FIG. 1 demonstrates an exemplary embodiment of the present screening methods for identification of aptamer sequences specific to a target that can be used in competition-based detection assays.

Target Binding Selections

In some embodiments, a pool of ssDNA molecules that substantially bind to a target molecule may be selected by a positive target-binding selection process comprising repeated target binding, partition, isolation and amplification of nucleic acid sequences using an input library comprising randomized ssDNA (single stranded DNA) molecules and a target material. Conventional aptamer selection processes may be used such as systematic evolution of ligands by exponential enrichment (SELEX), selected and amplified binding site (SAAB), cyclic amplification and selection of targets (CASTing), or the like. As a non-limiting example, a plurality of sequences that form ssDNA:target complexes may be identified by performing several rounds of positive Graphene Oxide (GO)-SELEX selection using an input ssDNA library comprising randomized single stranded DNA sequences and a target material (FIG. 1).

SELEX procedure generally involves a progressive selection, from a large library of double-stranded or single-stranded nucleic acids (DNAs, RNAs or DNA/RNA hybrids), of variable nucleic acid sequences that bind to a target of interest with high affinities and specificities by repeated rounds of target partition and amplification.

Each round of SELEX process consists of several steps including preparation of nucleic acid libraries, formation of nucleic acid-target complexes, separation between bound and unbound sequences, elution of aptamers, PCR amplification, and identification of aptamers specific to the target. Each round of selection enriches aptamer candidates from the nucleic acid library.

The input nucleic acid library may comprise a plurality of single-stranded DNA (ssDNA) molecules with randomized sequences. The ssDNA may be 50-150 nucleotides in length, for example, the ssDNA in the library is about 50 to 140 nucleotides in length, or about 50 to 130 nucleotides in length, or about 50 to 120 nucleotides in length, or about 50 to 100 nucleotides in length, or about 60 to 80 nucleotides in length, or about 70 to 90 nucleotides in length, or about 70-80 nucleotides in length. In some embodiments, the ssDNA in the library may be 60 nucleotides in length, or 61 nucleotides in length, or 62 nucleotides in length, or 63 nucleotides in length, or 64 nucleotides in length, or 65 nucleotides in length, or 66 nucleotides in length, or 67 nucleotides in length, or 68 nucleotides in length, or 69 nucleotides in length, or 70 nucleotides in length, or 71 nucleotides in length, or 72 nucleotides in length, or 73 nucleotides in length, or 74 nucleotides in length, or 75 nucleotides in length, or 76 nucleotides in length, or 77 nucleotides in length, or 78 nucleotides in length, or 79 nucleotides in length, or 80 nucleotides in length, or 81 nucleotides in length, or 82 nucleotides in length, or 83 nucleotides in length, or 84 nucleotides in length, or 85 nucleotides in length, or 86 nucleotides in length, or 87 nucleotides in length, or 88 nucleotides in length, or 89 nucleotides in length, or 90 nucleotides in length, or 91 nucleotides in length, or 92 nucleotides in length, or 93 nucleotides in length, or 94 nucleotides in length, or 95 nucleotides in length, or 96 nucleotides in length, or 97 nucleotides in length, or 98 nucleotides in length, or 99 nucleotides in length, or 100 nucleotides in length. Each ssDNA molecule in the library comprises a randomized nucleic acid sequence at the center flanked by a constant sequence at the 5′ end and a constant sequence at the 3′ end that serve as PCR primers, where the sequences of the primers are known, and the central randomized sequence may be 30 to 50 nucleotides in length. The randomized sequences can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.

As a non-limiting example, the input ssDNA molecule library may be generated by automated chemical synthesis on a DNA synthesizer.

As used herein, the “central randomized nucleic acid sequence” within an ss DNA may also be referred to as the “inner sequence” of the ssDNA.

In one preferred embodiment, the ssDNA molecules in the input library are 76 nucleotides in length, wherein a central randomized nucleic acid sequence with 30 nucleotides in length is flanked by two 23 nucleotides primers at the 5′ end and 3′-end of each ssDNA. As a non-limiting example, the 5′ end primer may comprise a nucleic acid sequence of 5′ TAGGGAAGAGAAGGACATATGAT3′ (SEQ ID NO. 1) and the 3′ end primer may comprise a nucleic acid sequence of 5′ TTGACTAGTACATGACCACTTGA 3′ (SEQ ID NO. 2).

As used herein, the term “primer” refers to a short nucleic acid which is capable of acting as a point of initiation of synthesis (e.g., PCR) when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded.

The input DNA library may be mixed with a target wherein the complexes are formed between the target and a plurality of ssDNA molecules present in the library. The target may be any molecule (e.g., nucleic acids, proteins, small molecules, sugars, toxins, biomarkers, cells and pathogens). In some embodiments, the target is a protein, such as an allergen protein or mixed allergen components of an allergen. The allergen may include, but is not limited to, a food allergen, an allergen from the environment such as plants, animals, microorganisms, air or water, and a medical allergen (i.e., any allergen found in a medicine or medical device).

Food allergens include, but are not limited to proteins in legumes such as peanuts, peas, lentils and beans, as well as the legume-related plant lupin, tree nuts such as almond, cashew, walnut, Brazil nut, filbert/hazelnut, pecan, pistachio, walnut, beechnut, butternut, chestnut, chinquapin nut, coconut, ginkgo nut, lychee nut, macadamia nut, nangai nut and pine nut, egg, fish, shellfish such as crab, crawfish, lobster, shrimp and prawns, mollusks such as clams, oysters, mussels and scallops, milk, soy, wheat, gluten, corn, meat such as beef, pork, lamb, mutton and chicken, gelatin, sulphite, seeds such as sesame, sunflower and poppy seeds, and spices such as coriander, garlic and mustard, fruits, vegetables such as celery, and rice. Some exemplary allergenic proteins from food allergens may include the parvalbumins in codfish, tropomyosin in crustaceans, arginine kinase and myosin light chain, casein, α-lactalbumin and 3 lactoglobulin in milk, and globulin or vicilin seed storage protein.

Other target molecules include, but are not limited to, pathogens from a pathogenic microorganism in a sample, such as bacteria, yeasts, fungi, spores, viruses and prions; disease proteins (e.g., biomarkers for diseases diagnosis and prognosis); pesticides and fertilizers remained in the environment; and toxins. Targets may include non-protein compounds such as minerals and small molecules (e.g., antibiotics).

In some embodiments, the steps for selecting an enriched pool of ssDNA molecules that substantially bind to the target material may comprise (i) contacting the input ssDNA library with the target material wherein complexes are formed between the target and a plurality of ssDNA molecules present in the input library; (ii) partitioning the complexes formed in step (i) from the unbound ssDNA molecules and isolating the ssDNA molecules in the complexes to produce a subset of ssDNA molecules for the target material and amplifying the isolated subset of ssDNA molecules; (iii) contacting the enriched subset of ssDNA molecules from step (ii) with the same target material wherein complexes are formed between the target and a second plurality of ssDNA molecules present in the enriched library to generate a second enriched subset group of ssDNA molecules; and (iv) optionally repeating steps of binding, partition, isolation and amplification (steps (i) to (iii)), one, two, three, four or more times as desired to yield highly specific, high affinity ssDNA molecules to the target molecule, thereby producing the enriched pool of ssDNA molecules that substantially bind to the target material (e.g., Pool 1 in Table 1).

In one embodiment, a graphene oxide (GO)-SELEX process modified from the general SELEX method is performed to select the target binding pool. As used herein, the terms “graphene,” “graphene oxide (GO),” “graphene oxide nanosheet” and “graphene nanosheet” mean two-dimensional carbon structures and are used interchangeably throughout the present specification. The exposed nucleobases in the ssDNA molecules can be absorbed to the surface of graphene oxide (Chen et al., J. Agric. Food Chem. 2014; 62, 10368-10374). Accordingly, when a graphene oxide (GO) solution is added to the mixture of a ssDNA library and a target material, GO can adsorb the ssDNA sequences that are not bound to a specific target, to its surface, and let the sequences bound to the target free. The unbound sequences and GO can then be removed, e.g., by centrifugation, while the ssDNA molecules that bind to a specific target are not absorbed to the surface of GO and then recovered and employed in the following selection process. This process can avoid the need to immobilize the target material as used in conventional SELEX.

The GO-SELEX process is inexpensive, fast, and simple. In a conventional SELEX, many expensive, less efficient and time-consuming methods such as chromatography, an affinity column, and the like, are used to separate nucleic acid molecules which are bound to a target material from nucleic acid molecules which are not bound to the target material. The GO-SELEX process is characterized in that the separation of binding ssDNA molecules from non-binding ssDNA molecules can be carried out simply by centrifugation even if the target material or the counter-target material is not specifically immobilized to a specific carrier (Nguyen et al., Chem. Commun. 2014, 50, 10513-10516; the contents of which are incorporated by reference herein in their entirety.)

After removing GO absorbed ssDNA molecules (e.g., by centrifugation), the target material may be removed from the collected DNA:target complexes. Methods for participating proteins in a solution well known in the art, for example, ethanol precipitation and strataclean resin may be used. As a non-limiting example, a strataclean resin may be added to the supernatant recovered after centrifugation. The target material bound to the strataclean resin can be removed by centrifugation. The target removal step may be repeated for two, three, four or more times. A supernatant containing the enriched ssDNA molecules that bind to the target material may be used for next target binding selection round (FIG. 1).

In some embodiments, the final concentration of ssDNA molecules that bind to the target material may be measured and compared to the initial concentration of the input ssDNA library. The ratio of the concentrations will be used to determine if another round of the GO-SELEX selection is needed. If the ratio is below 50%, another round of positive GO-SELEX process is carried out with the same condition. The same process is repeated until the recovery of ssDNA molecules that bind to the target material reaches to a satisfactory ratio, e.g., above 50% recovery. Rounds of partition and isolation are repeated until a desired goal is achieved, for example, two, three, four, five, six, seven, eight or more times with the same condition. In the most general case, selection is continued until no significant improvement in binding strength is achieved on repetition of the selection round.

The target binding pool of ssDNA molecules selected from the positive GO-SELEX process may be further amplified by performing a PCR using labeled primers, e.g., a biotinylated reverse primer and a fluorophore-labeled forward primer. In other aspects, the ssDNA molecules can be amplified by any other known method, such as sequencing the selected sequences and synthesizing them synthetically using an oligonucleotide synthesizer for the next round of binding and selection.

The fluorophore that is conjugated to the forward primer may be, but is not limited to, Cy5, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 658, Cyanine-3, Cyanine-5, fluorescein, Texas red, FITC (Fluorescein Isothiocyanate), rhodamine, or the like. In one preferred embodiment, the forward primer is conjugated with Cy5. In another embodiment, the forward primer is conjugated with Alexa Fluor 647.

After PCR amplification, the resulted double stranded DNA (dsDNA) molecules may be cleaned and further denatured to regenerate single stranded DNA (ssDNA) molecules. The biotinylated reverse primer allows for removal of the complementary strands to regenerate ssDNA molecules from the dsDNA molecules created during PCR amplification. As a non-limiting example, streptavidin coated magnetic beads may be added to the PCR product. The biotinylated complementary strands bind to the streptavidin coated magnetic beads. After denaturation of ssDNA molecules (e.g., addition of a base), the bound biotinylated complementary strands are separated and removed using a magnetic force. The desired ssDNA molecules with the fluorophore tags are collected. The fluorophore (e.g., Cy5 and Alexa Fluor 647) tagged ssDNA molecules that substantially bind to the target are used for next selection process.

The fluorophore (e.g., Cy5) tagged ssDNA molecules give several advantages in developing detection agents used in competition-based detection assays. The addition of Cy5 or other fluorescence markers to the ssDNA sequences at the beginning of aptamer selection can ensure that all aptamer candidates have proper secondary and tertiary structures when they are further developed as signaling polynucleotides (SPNs) used in detection assays. The addition of Cy5 or another fluorescence marker at the later stage may influence the secondary and tertiary structures of aptamer candidates. This modification can significantly reduce false hits during the selection.

As a non-limiting example, the GO-SELEX process to identify a pool of sequences that substantially bind to a target may comprise the steps of (i) mixing the input ssDNA library (e.g., Pool 0 in Table 1) with an allergen composition in a buffer solution; and these are induced to be bound to each other at normal temperature; (ii) adding a graphene oxide solution to the mixture of step (i) to remove ssDNA molecules which are not bound to the target; (iii) removing the target from the collected ssDNA molecules and amplifying the ssDNA molecules by performing a PCR using the PCR primers at the ends of the ssDNA molecules; and (iv) denaturing the double stranded PCT products and collecting ssDNA molecules labeled with fluorophore. Optionally, the positive GO-SELEX selection may be repeated for 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds. The ssDNA molecules in the input library comprise approximately 76 nucleotides in length, including a primer for PCR amplification at each end and about 30 nucleotides (the binding site) at its center (i.e., the inner sequence of an aptamer). The target material is an allergen material, particularly a food allergen, comprising one allergenic component, or a mixture of allergenic components from a single allergen. Food allergens may include but are not limited to proteins in legumes such as peanuts, peas, lentils and beans, tree nuts (e.g., almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut), wheat, milk, fish, egg white and sea food.

In some embodiments, the 5′ constant sequence (i.e., the 5′ primer) comprises a nucleic acid sequence of SEQ ID NO. 1 and the 3′ constant sequence (i.e., the 3′ primer) comprises a nucleic acid sequence of SEQ ID NO. 2.

On-Chip Target Binding and Competition Selections

The target binding pool of ssDNA molecules (e.g., Pool 1 in Table 1) may be further partitioned to select a subset of sequences that substantially bind to the target and compete with short oligonucleotides having sequences complementary to the ssDNA molecules. In some embodiments, this positive target binding selection may be performed using solid supports (e.g., glass or plastic chips) that are coated with short oligonucleotides comprising sequences complementary to the ssDNA molecules. By this process, families of nucleic acid sequences which can simultaneously bind to a target molecule and their complementary sequences may be subtracted from the pool. This additional positive selection process is tailored to differentiate ssDNA molecules that bind to the target and to the complementary sequences attached to a solid support (e.g., a glass or plastic chip).

The short oligonucleotide anchors may comprise sequences complementary to the constant sequences at the ends of the ssDNA molecules. The oligonucleotide anchors may comprise sequences complementary to either the 5′ end or 3′ end sequence of the aptamers. The complementary sequence contains about 5-25 nucleotides, or 5-18 nucleotides, or 6-20 nucleotides, or 8-20 nucleotides. For example, it may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides. In one preferred example, the complementary anchor sequence contains 5-15 nucleotides. The oligonucleotide anchor may be 100%, or 99%, or 98%, or 97%, or 96%, or 95%, or 94%, or 93%, or 92%, or 91%, or 90% complementary to the sequence of an ssDNA. The short complementary sequences are covalently linked to a solid support such as a glass chip, directly or through a linker.

The solid support on which the oligonucleotides are covalently attached may include, but is not limited to, a glass, a polymer support (e.g., see, U.S. Pat. No. 5,919,525), polyacrylamide gel, or plastic (e.g., a microwell plate), or a nylon membrane. The glass may be a polymer glass (e.g., acrylic glass, polycarbonate and polyethylene terephthalate), or a silicate glass (e.g., Pyrex glass, quartz and germanium-oxide glass), or a porous glass, etc., Polymers may include, but are not limited to, polyimide, photoresist, SU-8 negative photoresist, polydimethylsiloxane (PDMS), silicone elastomer PDMS and COC. In one preferred embodiment, the solid support is a glass chip.

Different technologies may be used for attaching the short complementary sequences to the solid support at determined sites. These methods are well-known in the pertinent art. For example, the oligonucleotides can be deposited on specific sites on the solid support as microdroplets by ink jet, or piezoelectric, or other similar methods. The solid support may be pre-treated to provide active attaching surfaces for oligonucleotides. In addition, the density of the attached oligonucleotides may be measured and controlled on the solid support.

In one embodiment, The on-chip target binding selection may comprise the steps: (i) mixing the target binding pool of ssDNA molecules (i.e., Pool 1) with the same target material in a buffer solution; and they are induced to be bound to each other at normal temperature; (ii) contacting the mixture of step (i) with a solid support of which the surface is covalently coated with short oligonucleotides comprising sequences complementary to the sequences of ssDNA molecules; (iii) collecting the ssDNA:target complexes that are not bound to the solid support (e.g., the flow-through) (FIG. 1); and (iv) removing the target material from the collected ssDNA:target complexes and collecting an enriched subset of ssDNA molecules. Optionally, the collected mixture in (iii) is again contacted with the solid support coated with the complementary oligonucleotides for two, three, four, five, six, seven, eight, or more times and the flow-through after the final incubation is processed to recover the ssDNA molecules from the complexes.

By this selection, a subset of ssDNA molecules in Pool 1 that are not bound to the target material will hybridize to the complimentary sequences covalently attached to the solid support and be removed from the pool. In addition, a subset of ssDNA molecules bound to the target material may also hybridize to the complimentary sequences attached to the solid support. These ssDNA molecules stay on the solid support and are subtracted from the collected ssDNA pool.

The collected ssDNA molecules from the final incubation are cleaned and separated from the target material as described herein. Similar to the positive GO-SELEX selection, the concentration of the recovered ssDNA molecules is measured and compared to the input pool (Pool 1). In some embodiments, the on-chip positive selection may be repeated for two, three, four, five, six, seven, eight or more rounds until the ratio of the recovered ssDNA molecules reaches to a desired recovery ratio (e.g., more than 50% from the input pool).

The ssDNA molecules are then amplified by performing PCR and single stranded DNA molecules are recovered as described in the positive GO-SELEX process. By this selection process, a pool of ssDNA molecules that bind to the target material but do not hybridize to the complementary sequences in the presence of the target material is selected (i.e. positive binding pool (Pool 2) in Table 1). The selection process mimics a condition used in a competition-based detection assay. The combination of regular SELEX (e.g., GO-SELEX) and on-chip positive target binding processes increases the specificity and affinity of aptamers.

On-Chip Negative (Counter) Selections

The positive pool of ssDNA molecules (Pool 2) containing aptamer candidates that bind to the target material in the presence of the complementary sequences may be further screened to isolate ssDNA molecules that do not bind to the complementary sequences even when the sequences are free, and sequences that substantially bind to counter target materials in addition to the target of interest. In some embodiments, these non-specific ssDNA sequences may be isolated by counter selection processes using solid supports (e.g., glass chips) precoated with short oligonucleotides comprising sequences complementary to the ssDNA molecules.

In some embodiments, an on-chip non-binding counter selection is performed to identify ssDNA molecules that do not hybridize to their complementary sequences in the pool even when they are free. A DNA solution comprising the positive pool of ssDNA molecules (Pool 2), without addition of the target material, is directly incubated with a solid support (e.g., a glass chip) that is precoated with short oligonucleotides comprising sequences complementary to the aptamers in the pool. After incubation, the DNA solution including the unbound ssDNA molecules is collected (i.e. the flow-through) (FIG. 1). The flow-through DNA solution is incubated again with the solid support (e.g., a glass chip) that is precoated with short complementary oligonucleotides and a second flow-through is collected. The incubation step may be repeated two, three, four, five, six, seven, eight or more times, preferably eight times.

In one preferred embodiment, the on-chip non-binding counter process may comprise the steps: (i) preparing a DNA solution comprising the positive binding pool of ssDNA molecules (Pool 2); (ii) contacting the DNA solution with a solid support coated with short oligonucleotides comprising sequences complementary to the ssDNA molecules; (iii) collecting the ssDNA solution after incubation; and (iv) contacting the collected solution again with a new solid support coated with the complementary sequences. These steps may be repeated for two, three, four, five, six, seven or eight rounds and the collected ssDNA solution from the final incubation will be cleaned and amplified for sequencing. In one embodiment, these steps are repeated for eight rounds and the collected ssDNA solution from the final incubation are cleaned and amplified for sequencing.

This on-chip non-binding counter selection creates a non-binding pool of ssDNA molecules (i.e., Pool 3 in Table 1) including ssDNA molecules that cannot hybridize to the complementary sequences even in the absence of the target material.

In other embodiments, an on-chip counter binding selection is performed to isolate any sequences that can bind to non-specific counter targets from the positive binding pool of ssDNA molecules (Pool 2 in Table 1). This counter selection process improves the target specificity of selected ssDNA molecules by eliminating nucleic acid sequences with cross-reactivity to one or more non-target molecules (e.g., counter targets).

In one preferred embodiment, the on-chip counter selection process may comprise the steps of (i) preparing a ssDNA solution comprising the positive binding pool of ssDNA molecules (Pool 2) and incubating the ssDNA solution with a counter target or a mixture of counter targets; (ii) contacting the mixture of step (i) with a solid support that is coated with short oligonucleotides comprising sequences complementary to the ssDNA molecules; (iii) collecting the ssDNA/counter target complexes after step (ii) (i.e. the flow-through) (FIG. 1); and (iv) contacting the collected solution in step (iii) to a solid support that is coated with complementary oligonucleotides. The incubation and collection steps may be repeated for two, three, four, five, six, seven, eight or more rounds, preferably eight rounds. The collected solution after the final incubation step will be cleaned and/or amplified for sequencing.

In some embodiments, the on-chip counter binding selection may be repeated for as many counter targets as desired, beginning each time with the same pool of ssDNA molecules from the positive binding pool (i.e., Pool 2 in Table 1). In some alternative embodiments, multiple counter targets can be run within the same round in parallel.

By this on-chip counter selection process, ssDNA sequences with cross-specificity towards undesirable related proteins (counter target material) are removed from the positive binding pool. This counter selection process creates a pool of ssDNA molecules (i.e., Pool 4 in Table 1) including the ssDNA molecules that can cross react with a counter target or several counter targets.

As non-limiting examples, the counter targets may be allergen proteins in the same family, including allergen proteins from different sources that can be attributed to these structurally related allergen families, e.g., prolamins family including seed storage proteins (e.g., Sec c 20 in Rye; Tri a 19 in wheat and Tri a 36 in wheat), non-specific lipid transfer proteins family (e.g., Act d 10 in Kiwi, Api g 2 in celery, Ara h 9 in peanut, Cas s 8 in chestnut, Cor a 8 in hazelnut, Jug r 3 in walnut, Lyc e 3 in tomato, Mus a 3 in banana, and Pru du 3 in almond), 2S albumins family including seed storage proteins (e.g., Ana o 3 in cashew nut, Ara h 2 in peanut, Ber e 1 in Brazil nut, Fag e 2 in buckwheat, Gly m 8 in soybean, Jug r 1 in walnut, Ses i 1 in sesame, and Sin a 1 in mustard), Bet VI family including pathogenesis related proteins (e.g., Api g 1/celery, Ara h 8/peanut, Cor a 1/hazelnut, Dau c 1/carrot, Gly m 4/soybean, Mal d 1/apple, and Pru p 1/peach), 7S (vicilin-like) globulins family (e.g., Ana o 1/cashew nut; Ara h 1/peanut; Gly m 5/soybean; Jug r 2/walnut; Pis v 3/pistachio), 11S (legumin-like) globulins family (e.g., Ana o 2/cashew nut; Ara h 3/peanut; Ber e 2/Brazil nut; Cor a 9/hazelnut; Gly m 6/soybean; Jug r 4/walnut; Pru du 6/almond), Cysteine protease C1 family (e.g., Act d 1/kiwi; Gly m Bd 30K/soybean), Profilins family including actin binding proteins (e.g., Act d 9/kiwi; Api g 4/celery; Ara h 5/peanut; Cuc m 2/melon; Dau c 4/carrot; Gly m 3/soybean; Lyc e 1/tomato; Mus a 1/banana; Ory s 12/rice; Pru av 4/cherry; Pru du 4/almond; Pru p 4/peach and Tri a 12/wheat), tropomyosin family including actin binding proteins in muscle (e.g., Pen m 1/shrimp), parvalbumin family including muscle proteins (e.g., Cyp c 1/carp; Gad c 1/cod; Ran e 2/frog; Sal s 1/salmon; Seb m 1/redfish; Xip g 1/swordfish), caseins family including mammalian milk proteins (e.g., Bos d 8-Bos d 12/cow's milk), transferrin family including sulfur-rich ion-binding glycoproteins from milk and hen's egg white (e.g., Bos d Lactoferrin/cow's milk; Gal d 3/hen's egg), serpins family including Serine protease inhibitors (e.g., Gal d 2/hen's egg), Arginine kinases family including Adenosine triphosphate:guanido phosphotransferases (e.g., Pen m 2/shrimp), Lipocalins family including carrier proteins (e.g., Bos d 5/cow's milk), Ovomucoids family including Kazal inhibitors (e.g., Gal d 1/hen's egg), Lysozyme family (e.g., Bos d 4/cow's milk; Gal d 4/hen's egg), and Albumins family including Serum albumins (e.g., Bos d 6/cow's milk; Gal d 5/hen's egg).

Deep Sequencing

In accordance with the present screening method, the ssDNA pools (e.g., Pool 1, Pool 2, Pool 3 and Pool 4 in Table 1) from each selection may be cleaned, amplified and sequenced. In one embodiment, the method comprises an amplification of the individual ssDNA molecules using a polymerase chain reaction (PCR). The sequences within each pool are identified using deep sequencing. In some embodiments, the ssDNA molecules in the target binding pool (i.e., Pool 1) are amplified and sequenced. In parallel, an artifact library may be made by amplifying the input ssDNA library and the sequences in this artifact library are sequenced (See, e.g., the flow-chart of FIG. 1). The artifact library may be made from repeating the PCR amplification and strand separation steps for the same number of rounds for the positive GO-SELEX selection (FIG. 1). These sequences, resulted from over amplification by PCR, are removed from the target binding pool (Pool 2 in Table 1).

The ssDNA molecules in the positive binding pool from the final round of the on-chip target binding selection (e.g., Pool 2 in Table 1) may be sequenced. The ssDNA molecules within this pool contain ssDNA sequences that preferentially bind to their target in the presence of their complementary sequences.

The non-specific ssDNA molecules from the final round of the on-chip non-binding counter selection and from the final round of the on-chip counter selection (e.g., Pools 3 and 4 in Table 1) may be sequenced. The ssDNA molecules within these pools contain ssDNA sequences that fail to hybridize to the complementary sequences even in the absence of the target material and sequences with cross-specificity to other counter targets.

The ssDNA sequences from each pool may be barcoded for identity. Following barcoding, ssDNA molecules from each pool may be pooled together and run deep sequencing in a single lane on the Illumina MiSeq System.

TABLE 1 Summary of Selection rounds DNA pool Description Input library A library of random synthesized single Input (Pool 0) stranded DNA molecules comprising a central sequences randomized region and two primer regions at both ends. Target A sub-pool of ssDNA molecules that positively Sequenced binding pool bind to the target of interest selected by (Pool 1) the GO-SELEX process Positive pool A sub-pool of ssDNA molecules that Sequenced (Pool 2) preferentially bind to the target of interest to various short complementary sequences, selected by the on-chip positive target binding process Non-binder A sub-pool of ssDNA molecules that do not Sequenced pool bind to various short complementary sequences (Pool 3) in the absence of the target selected from the on-chip non-binding counter selection Counter A sub-pool of ssDNA molecules that bind to Sequenced binding pool various counter targets in addition to binding (Pool 4) to the target of interest, selected by the on- chip counter selection Aptamer a final subset of ssDNA molecules after selected candidate extracting the sequences in Pool 3 and Pool pool 4 from Pool 2 (Pool 5)

Data Analysis and Bioinformatics

After sequencing and barcoding of the ssDNA sequences in each pool, and running the deep sequencing, the data are analyzed using any available bioinformatics tools. In some embodiments, heat maps are generated for each individual pool, which represent the frequencies of ssDNA sequences in each pool, by using a local occurrence of the open-source bioinformatics tool Galaxy (Thiel and Giangrande, Methods 2016, 97, 3-10; the contents of which are incorporated herein by reference in their entirety).

Potential aptamer hits are selected by analyzing the over-expressed sequences in each pool using the heat maps for sequences in each pool. Essentially, the heat maps of the ssDNA molecules from the non-binding pool (Pool 3) and the counter binding pool (Pool 4) are subtracted from the heat maps of the ssDNA molecules from the positive binding pool (Pool 2). The final data represent a pool of potential aptamer hits with characteristics including: (i) binding to the target protein with high specificity and affinity, (ii) hybridizing to their short complementary sequences only in the absence of the target but not binding to the short complementary sequences in the presence of the target; and (iii) no cross-reactivity to non-specific counter targets. These characteristics of the aptamer candidates make them suitable for target detection in a sample, e.g., in competition-based assays.

From the final pool of potential aptamer hits (e.g., Pool 5 in Table 1), a sequence family tree may be constructed to show the similarities between different aptamer sequences. Multiple sequences from various branches of the family tree structure can be selected and folded using expected assay conditions. The secondary and tertiary structures will be assessed, and those sequences that show multiple well-defined structures are selected for synthesis and further evaluation. The structures or motifs may include hairpin loops, symmetric and asymmetric bulges, pseudoknots and myriad combinations of the same. The equilibrium dissociation constant (K_(d)), and other parameters of the selected aptamers will be measured.

In accordance with the present disclosure, the selection method may further comprise the steps of (i) amplifying all the sequences in the first, second, third and fourth sub-pools, and barcoding each sequence from each pool; (ii) pooling the sequences from each sub-pool together and running sequencing together; (iii) analyzing the data from (ii) and separating each sequence data into the original sub-pool according to the barcode information; (iv) generating heat maps for each individual sub-pool that represent the frequencies of each sequence in the pool; and (v) subtracting the sequences in the heat maps of the third sub-pool and the fourth sub-pool from the heat maps of the second sub-pool, wherein the final pool of sequences are candidate aptamers that specifically bind to the target of interest and preferentially bind to the target of the interest in competing the binding of various short complementary sequences.

In accordance with the present disclosure, sequences that specifically bind to peanut, tree nuts including almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut, gluten, milk allergens whey and casein are selected. Aptamer sequences that bind to all nuts are also selected. As used here, the term “all nuts” refers to peanut and the tree nuts including almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut. A selected aptamer sequence that is specific to “all nuts” can bind to any of the nuts (i.e., peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut), e.g., one, two, three, four, five, six, seven or eight nuts present in samples.

In some embodiments, the sequences that specifically bind to peanut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 3 to 1002.

In some embodiments, the sequences that specifically bind to almond comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 4003 to 5002.

In some embodiments, the sequences that specifically bind to brazil nut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 8003 to 9002.

In some embodiments, the sequences that specifically bind to cashew comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 12003 to 13002.

In some embodiments, the sequences that specifically bind to hazelnut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 16003 to 17002.

In some embodiments, the sequences that specifically bind to pecan comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 20003 to 21002.

In some embodiments, the sequences that specifically bind to pistachio comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 24003 to 25002.

In some embodiments, the sequences that specifically bind to walnut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 28003 to 29002.

In some embodiments, the sequences that specifically bind to all nuts comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 32003 to 33002.

In some embodiments, the sequences that specifically bind to gluten comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 40003 to 41002.

In some embodiments, the sequences that specifically bind to whey comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 44003 to 45002.

In some embodiments, the sequences that specifically bind to casein comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NO. 48003 to 49002.

Multiple SELEX Selections

In some embodiments, the present selection method may be modified to identify aptamer sequences that bind to multiple targets. The multiple target selection process provides an efficient method for identifying the best binding aptamers to a group of targets.

Many allergens, particularly food allergens, are composed of multiple allergenic components. These components may induce component specific IgE in a person's body. Some people are allergic to only one specific component but not the other components of the same allergen. Some people are allergic to all the components of an allergen. For example, milk includes two primary allergenic components: the whey proteins (alpha-lactalbumin and beta-lactoglobulin) and caseins. A person who is allergic to milk, may be allergic to only whey or casein, or to both whey and casein. In this context, an aptamer ligand that binds to the whey proteins only, or caseins only, or both the whey proteins and caseins, may be desirable to milk allergy.

In some embodiments, the present screening methods may be modified for selecting aptamers that can bind to the multiple components of an allergen.

As a non-limiting example, two, three, four or more rounds of the positive GO-SELEX selection are performed using the whole allergen material as the target material (e.g., the whole milk including casein and the whey proteins). The ssDNA molecules selected from this process (Pool 1) include a mixture of ssDNA sequences that bind to any of the various components of milk (e.g., caseins and the whey proteins). These milk binding sequences are used to run two parallel selection processes: a selection process for casein only and a selection process for the whey protein only. The two selection processes are performed as previously described for a single target. Importantly, during the counter selection process, a separate selection will be performed using only the other component as the counter target. That is, in the selection for the aptamer sequences that specifically bind to casein, a whey protein is used as the counter target in the counter selection process, while casein is used as the counter target for selecting aptamer sequences that specifically bind to a whey protein.

Following similar procedures, the various sub-pools of ssDNA molecules will be barcoded and submitted for deep sequencing. The casein and whey samples will be pooled separately from one another and run in separate lanes during deep sequencing. The bioinformatic analysis of the sequencing data will reveal the pool of aptamer hits for the target alone. In order to find an aptamer sequence that binds both casein and whey, overlaps between the special counter rounds may be collected.

In another example, a mixture of all nuts may be used as target materials, sequences that can recognize all nuts may be selected by the present methods. The selected sequences may bind to any nut, and the combinations of any nuts present in a test sample.

Aptamers, Signaling Polynucleotides (SPNs) and Detection Sensors

In another aspect of the disclosure, aptamers that specifically bind to allergen targets, signaling polynucleotides (SPNs) derived from the selected aptamers, and detection sensors comprising these aptamers and SPNs are provided. An aptamer that binds to a target allergen with high specificity and affinity may not hybridize to the short complementary sequence in the presence of the target allergen, and demonstrates little or no cross-specificity to any counter target.

A SPN may be derived from an aptamer sequence selected by the present method. The SPN may further comprise additional nucleotides at one end or both ends of the aptamer sequence. The sequence may be further modified to change its secondary and/or tertiary structures to make it more stable, to increase the binding affinity and/or specificity, or to add a fluorescence marker, or to be modified to comprise one or more conjugates.

Detection sensors comprising selected aptamers and SPNs are provided. In some embodiments, the detection sensor may include a SPN, a solid support and a short oligonucleotide comprising a nucleic acid sequence complementary to the SPN, wherein the oligonucleotide is covalently anchored to the solid support by one of the ends, directly or through a linker (e.g., a 6 carbon atom arm). The SPN comprises an inner sequence that specifically binds to a target of interest and it hybridizes to the complementary oligonucleotide when it is not bound to the target of interest. In one example, the short complementary sequences and the target of interest will compete binding to the SPN. In this competitive assay, for example, the SPN can either bind to the short complementary sequences attached on the solid support or a target of interest in a sample. Under conditions sufficient to allow the target of interest in the sample to compete with the short complementary sequences attached on the solid support, the SPN:target complexes can be detected and measured.

In accordance with the present disclosure, aptamer sequences that specifically bind to peanut, tree nuts including almond, brazil nut, cashew, hazelnut, pecan, pistachio and walnut, gluten, milk allergens whey and casein are selected. Aptamer sequences that bind to all nuts are also selected.

In some embodiments, the sequences that specifically bind to peanut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs.3 to 1002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO.1. Accordingly, the aptamer that specifically binds to peanut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs.1003 to 2002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO.2. Accordingly, the aptamer that specifically binds to peanut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 2003 to 3002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to peanut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 3003 to 4002. In one embodiment, the aptamer of the present disclosure that specifically binds to peanut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 3 to 4002 listed in Table 2, or variant thereof.

TABLE 2 Aptamer sequences against peanut Aptamer sequence 5′ sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′ sequence 3′ sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 3 1003 2003 3003 4 1004 2004 3004 5 1005 2005 3005 6 1006 2006 3006 7 1007 2007 3007 8 1008 2008 3008 9 1009 2009 3009 10 1010 2010 3010 11 1011 2011 3011 12 1012 2012 3012 13 1013 2013 3013 14 1014 2014 3014 15 1015 2015 3015 16 1016 2016 3016 17 1017 2017 3017 18 1018 2018 3018 19 1019 2019 3019 20 1020 2020 3020 21 1021 2021 3021 22 1022 2022 3022 23 1023 2023 3023 24 1024 2024 3024 25 1025 2025 3025 26 1026 2026 3026 27 1027 2027 3027 28 1028 2028 3028 29 1029 2029 3029 30 1030 2030 3030 31 1031 2031 3031 32 1032 2032 3032 33 1033 2033 3033 34 1034 2034 3034 35 1035 2035 3035 36 1036 2036 3036 37 1037 2037 3037 38 1038 2038 3038 39 1039 2039 3039 40 1040 2040 3040 41 1041 2041 3041 42 1042 2042 3042 43 1043 2043 3043 44 1044 2044 3044 45 1045 2045 3045 46 1046 2046 3046 47 1047 2047 3047 48 1048 2048 3048 49 1049 2049 3049 50 1050 2050 3050 51 1051 2051 3051 52 1052 2052 3052 53 1053 2053 3053 54 1054 2054 3054 55 1055 2055 3055 56 1056 2056 3056 57 1057 2057 3057 58 1058 2058 3058 59 1059 2059 3059 60 1060 2060 3060 61 1061 2061 3061 62 1062 2062 3062 63 1063 2063 3063 64 1064 2064 3064 65 1065 2065 3065 66 1066 2066 3066 67 1067 2067 3067 68 1068 2068 3068 69 1069 2069 3069 70 1070 2070 3070 71 1071 2071 3071 72 1072 2072 3072 73 1073 2073 3073 74 1074 2074 3074 75 1075 2075 3075 76 1076 2076 3076 77 1077 2077 3077 78 1078 2078 3078 79 1079 2079 3079 80 1080 2080 3080 81 1081 2081 3081 82 1082 2082 3082 83 1083 2083 3083 84 1084 2084 3084 85 1085 2085 3085 86 1086 2086 3086 87 1087 2087 3087 88 1088 2088 3088 89 1089 2089 3089 90 1090 2090 3090 91 1091 2091 3091 92 1092 2092 3092 93 1093 2093 3093 94 1094 2094 3094 95 1095 2095 3095 96 1096 2096 3096 97 1097 2097 3097 98 1098 2098 3098 99 1099 2099 3099 100 1100 2100 3100 101 1101 2101 3101 102 1102 2102 3102 103 1103 2103 3103 104 1104 2104 3104 105 1105 2105 3105 106 1106 2106 3106 107 1107 2107 3107 108 1108 2108 3108 109 1109 2109 3109 110 1110 2110 3110 111 1111 2111 3111 112 1112 2112 3112 113 1113 2113 3113 114 1114 2114 3114 115 1115 2115 3115 116 1116 2116 3116 117 1117 2117 3117 118 1118 2118 3118 119 1119 2119 3119 120 1120 2120 3120 121 1121 2121 3121 122 1122 2122 3122 123 1123 2123 3123 124 1124 2124 3124 125 1125 2125 3125 126 1126 2126 3126 127 1127 2127 3127 128 1128 2128 3128 129 1129 2129 3129 130 1130 2130 3130 131 1131 2131 3131 132 1132 2132 3132 133 1133 2133 3133 134 1134 2134 3134 135 1135 2135 3135 136 1136 2136 3136 137 1137 2137 3137 138 1138 2138 3138 139 1139 2139 3139 140 1140 2140 3140 141 1141 2141 3141 142 1142 2142 3142 143 1143 2143 3143 144 1144 2144 3144 145 1145 2145 3145 146 1146 2146 3146 147 1147 2147 3147 148 1148 2148 3148 149 1149 2149 3149 150 1150 2150 3150 151 1151 2151 3151 152 1152 2152 3152 153 1153 2153 3153 154 1154 2154 3154 155 1155 2155 3155 156 1156 2156 3156 157 1157 2157 3157 158 1158 2158 3158 159 1159 2159 3159 160 1160 2160 3160 161 1161 2161 3161 162 1162 2162 3162 163 1163 2163 3163 164 1164 2164 3164 165 1165 2165 3165 166 1166 2166 3166 167 1167 2167 3167 168 1168 2168 3168 169 1169 2169 3169 170 1170 2170 3170 171 1171 2171 3171 172 1172 2172 3172 173 1173 2173 3173 174 1174 2174 3174 175 1175 2175 3175 176 1176 2176 3176 177 1177 2177 3177 178 1178 2178 3178 179 1179 2179 3179 180 1180 2180 3180 181 1181 2181 3181 182 1182 2182 3182 183 1183 2183 3183 184 1184 2184 3184 185 1185 2185 3185 186 1186 2186 3186 187 1187 2187 3187 188 1188 2188 3188 189 1189 2189 3189 190 1190 2190 3190 191 1191 2191 3191 192 1192 2192 3192 193 1193 2193 3193 194 1194 2194 3194 195 1195 2195 3195 196 1196 2196 3196 197 1197 2197 3197 198 1198 2198 3198 199 1199 2199 3199 200 1200 2200 3200 201 1201 2201 3201 202 1202 2202 3202 203 1203 2203 3203 204 1204 2204 3204 205 1205 2205 3205 206 1206 2206 3206 207 1207 2207 3207 208 1208 2208 3208 209 1209 2209 3209 210 1210 2210 3210 211 1211 2211 3211 212 1212 2212 3212 213 1213 2213 3213 214 1214 2214 3214 215 1215 2215 3215 216 1216 2216 3216 217 1217 2217 3217 218 1218 2218 3218 219 1219 2219 3219 220 1220 2220 3220 221 1221 2221 3221 222 1222 2222 3222 223 1223 2223 3223 224 1224 2224 3224 225 1225 2225 3225 226 1226 2226 3226 227 1227 2227 3227 228 1228 2228 3228 229 1229 2229 3229 230 1230 2230 3230 231 1231 2231 3231 232 1232 2232 3232 233 1233 2233 3233 234 1234 2234 3234 235 1235 2235 3235 236 1236 2236 3236 237 1237 2237 3237 238 1238 2238 3238 239 1239 2239 3239 240 1240 2240 3240 241 1241 2241 3241 242 1242 2242 3242 243 1243 2243 3243 244 1244 2244 3244 245 1245 2245 3245 246 1246 2246 3246 247 1247 2247 3247 248 1248 2248 3248 249 1249 2249 3249 250 1250 2250 3250 251 1251 2251 3251 252 1252 2252 3252 253 1253 2253 3253 254 1254 2254 3254 255 1255 2255 3255 256 1256 2256 3256 257 1257 2257 3257 258 1258 2258 3258 259 1259 2259 3259 260 1260 2260 3260 261 1261 2261 3261 262 1262 2262 3262 263 1263 2263 3263 264 1264 2264 3264 265 1265 2265 3265 266 1266 2266 3266 267 1267 2267 3267 268 1268 2268 3268 269 1269 2269 3269 270 1270 2270 3270 271 1271 2271 3271 272 1272 2272 3272 273 1273 2273 3273 274 1274 2274 3274 275 1275 2275 3275 276 1276 2276 3276 277 1277 2277 3277 278 1278 2278 3278 279 1279 2279 3279 280 1280 2280 3280 281 1281 2281 3281 282 1282 2282 3282 283 1283 2283 3283 284 1284 2284 3284 285 1285 2285 3285 286 1286 2286 3286 287 1287 2287 3287 288 1288 2288 3288 289 1289 2289 3289 290 1290 2290 3290 291 1291 2291 3291 292 1292 2292 3292 293 1293 2293 3293 294 1294 2294 3294 295 1295 2295 3295 296 1296 2296 3296 297 1297 2297 3297 298 1298 2298 3298 299 1299 2299 3299 300 1300 2300 3300 301 1301 2301 3301 302 1302 2302 3302 303 1303 2303 3303 304 1304 2304 3304 305 1305 2305 3305 306 1306 2306 3306 307 1307 2307 3307 308 1308 2308 3308 309 1309 2309 3309 310 1310 2310 3310 311 1311 2311 3311 312 1312 2312 3312 313 1313 2313 3313 314 1314 2314 3314 315 1315 2315 3315 316 1316 2316 3316 317 1317 2317 3317 318 1318 2318 3318 319 1319 2319 3319 320 1320 2320 3320 321 1321 2321 3321 322 1322 2322 3322 323 1323 2323 3323 324 1324 2324 3324 325 1325 2325 3325 326 1326 2326 3326 327 1327 2327 3327 328 1328 2328 3328 329 1329 2329 3329 330 1330 2330 3330 331 1331 2331 3331 332 1332 2332 3332 333 1333 2333 3333 334 1334 2334 3334 335 1335 2335 3335 336 1336 2336 3336 337 1337 2337 3337 338 1338 2338 3338 339 1339 2339 3339 340 1340 2340 3340 341 1341 2341 3341 342 1342 2342 3342 343 1343 2343 3343 344 1344 2344 3344 345 1345 2345 3345 346 1346 2346 3346 347 1347 2347 3347 348 1348 2348 3348 349 1349 2349 3349 350 1350 2350 3350 351 1351 2351 3351 352 1352 2352 3352 353 1353 2353 3353 354 1354 2354 3354 355 1355 2355 3355 356 1356 2356 3356 357 1357 2357 3357 358 1358 2358 3358 359 1359 2359 3359 360 1360 2360 3360 361 1361 2361 3361 362 1362 2362 3362 363 1363 2363 3363 364 1364 2364 3364 365 1365 2365 3365 366 1366 2366 3366 367 1367 2367 3367 368 1368 2368 3368 369 1369 2369 3369 370 1370 2370 3370 371 1371 2371 3371 372 1372 2372 3372 373 1373 2373 3373 374 1374 2374 3374 375 1375 2375 3375 376 1376 2376 3376 377 1377 2377 3377 378 1378 2378 3378 379 1379 2379 3379 380 1380 2380 3380 381 1381 2381 3381 382 1382 2382 3382 383 1383 2383 3383 384 1384 2384 3384 385 1385 2385 3385 386 1386 2386 3386 387 1387 2387 3387 388 1388 2388 3388 389 1389 2389 3389 390 1390 2390 3390 391 1391 2391 3391 392 1392 2392 3392 393 1393 2393 3393 394 1394 2394 3394 395 1395 2395 3395 396 1396 2396 3396 397 1397 2397 3397 398 1398 2398 3398 399 1399 2399 3399 400 1400 2400 3400 401 1401 2401 3401 402 1402 2402 3402 403 1403 2403 3403 404 1404 2404 3404 405 1405 2405 3405 406 1406 2406 3406 407 1407 2407 3407 408 1408 2408 3408 409 1409 2409 3409 410 1410 2410 3410 411 1411 2411 3411 412 1412 2412 3412 413 1413 2413 3413 414 1414 2414 3414 415 1415 2415 3415 416 1416 2416 3416 417 1417 2417 3417 418 1418 2418 3418 419 1419 2419 3419 420 1420 2420 3420 421 1421 2421 3421 422 1422 2422 3422 423 1423 2423 3423 424 1424 2424 3424 425 1425 2425 3425 426 1426 2426 3426 427 1427 2427 3427 428 1428 2428 3428 429 1429 2429 3429 430 1430 2430 3430 431 1431 2431 3431 432 1432 2432 3432 433 1433 2433 3433 434 1434 2434 3434 435 1435 2435 3435 436 1436 2436 3436 437 1437 2437 3437 438 1438 2438 3438 439 1439 2439 3439 440 1440 2440 3440 441 1441 2441 3441 442 1442 2442 3442 443 1443 2443 3443 444 1444 2444 3444 445 1445 2445 3445 446 1446 2446 3446 447 1447 2447 3447 448 1448 2448 3448 449 1449 2449 3449 450 1450 2450 3450 451 1451 2451 3451 452 1452 2452 3452 453 1453 2453 3453 454 1454 2454 3454 455 1455 2455 3455 456 1456 2456 3456 457 1457 2457 3457 458 1458 2458 3458 459 1459 2459 3459 460 1460 2460 3460 461 1461 2461 3461 462 1462 2462 3462 463 1463 2463 3463 464 1464 2464 3464 465 1465 2465 3465 466 1466 2466 3466 467 1467 2467 3467 468 1468 2468 3468 469 1469 2469 3469 470 1470 2470 3470 471 1471 2471 3471 472 1472 2472 3472 473 1473 2473 3473 474 1474 2474 3474 475 1475 2475 3475 476 1476 2476 3476 477 1477 2477 3477 478 1478 2478 3478 479 1479 2479 3479 480 1480 2480 3480 481 1481 2481 3481 482 1482 2482 3482 483 1483 2483 3483 484 1484 2484 3484 485 1485 2485 3485 486 1486 2486 3486 487 1487 2487 3487 488 1488 2488 3488 489 1489 2489 3489 490 1490 2490 3490 491 1491 2491 3491 492 1492 2492 3492 493 1493 2493 3493 494 1494 2494 3494 495 1495 2495 3495 496 1496 2496 3496 497 1497 2497 3497 498 1498 2498 3498 499 1499 2499 3499 500 1500 2500 3500 501 1501 2501 3501 502 1502 2502 3502 503 1503 2503 3503 504 1504 2504 3504 505 1505 2505 3505 506 1506 2506 3506 507 1507 2507 3507 508 1508 2508 3508 509 1509 2509 3509 510 1510 2510 3510 511 1511 2511 3511 512 1512 2512 3512 513 1513 2513 3513 514 1514 2514 3514 515 1515 2515 3515 516 1516 2516 3516 517 1517 2517 3517 518 1518 2518 3518 519 1519 2519 3519 520 1520 2520 3520 521 1521 2521 3521 522 1522 2522 3522 523 1523 2523 3523 524 1524 2524 3524 525 1525 2525 3525 526 1526 2526 3526 527 1527 2527 3527 528 1528 2528 3528 529 1529 2529 3529 530 1530 2530 3530 531 1531 2531 3531 532 1532 2532 3532 533 1533 2533 3533 534 1534 2534 3534 535 1535 2535 3535 536 1536 2536 3536 537 1537 2537 3537 538 1538 2538 3538 539 1539 2539 3539 540 1540 2540 3540 541 1541 2541 3541 542 1542 2542 3542 543 1543 2543 3543 544 1544 2544 3544 545 1545 2545 3545 546 1546 2546 3546 547 1547 2547 3547 548 1548 2548 3548 549 1549 2549 3549 550 1550 2550 3550 551 1551 2551 3551 552 1552 2552 3552 553 1553 2553 3553 554 1554 2554 3554 555 1555 2555 3555 556 1556 2556 3556 557 1557 2557 3557 558 1558 2558 3558 559 1559 2559 3559 560 1560 2560 3560 561 1561 2561 3561 562 1562 2562 3562 563 1563 2563 3563 564 1564 2564 3564 565 1565 2565 3565 566 1566 2566 3566 567 1567 2567 3567 568 1568 2568 3568 569 1569 2569 3569 570 1570 2570 3570 571 1571 2571 3571 572 1572 2572 3572 573 1573 2573 3573 574 1574 2574 3574 575 1575 2575 3575 576 1576 2576 3576 577 1577 2577 3577 578 1578 2578 3578 579 1579 2579 3579 580 1580 2580 3580 581 1581 2581 3581 582 1582 2582 3582 583 1583 2583 3583 584 1584 2584 3584 585 1585 2585 3585 586 1586 2586 3586 587 1587 2587 3587 588 1588 2588 3588 589 1589 2589 3589 590 1590 2590 3590 591 1591 2591 3591 592 1592 2592 3592 593 1593 2593 3593 594 1594 2594 3594 595 1595 2595 3595 596 1596 2596 3596 597 1597 2597 3597 598 1598 2598 3598 599 1599 2599 3599 600 1600 2600 3600 601 1601 2601 3601 602 1602 2602 3602 603 1603 2603 3603 604 1604 2604 3604 605 1605 2605 3605 606 1606 2606 3606 607 1607 2607 3607 608 1608 2608 3608 609 1609 2609 3609 610 1610 2610 3610 611 1611 2611 3611 612 1612 2612 3612 613 1613 2613 3613 614 1614 2614 3614 615 1615 2615 3615 616 1616 2616 3616 617 1617 2617 3617 618 1618 2618 3618 619 1619 2619 3619 620 1620 2620 3620 621 1621 2621 3621 622 1622 2622 3622 623 1623 2623 3623 624 1624 2624 3624 625 1625 2625 3625 626 1626 2626 3626 627 1627 2627 3627 628 1628 2628 3628 629 1629 2629 3629 630 1630 2630 3630 631 1631 2631 3631 632 1632 2632 3632 633 1633 2633 3633 634 1634 2634 3634 635 1635 2635 3635 636 1636 2636 3636 637 1637 2637 3637 638 1638 2638 3638 639 1639 2639 3639 640 1640 2640 3640 641 1641 2641 3641 642 1642 2642 3642 643 1643 2643 3643 644 1644 2644 3644 645 1645 2645 3645 646 1646 2646 3646 647 1647 2647 3647 648 1648 2648 3648 649 1649 2649 3649 650 1650 2650 3650 651 1651 2651 3651 652 1652 2652 3652 653 1653 2653 3653 654 1654 2654 3654 655 1655 2655 3655 656 1656 2656 3656 657 1657 2657 3657 658 1658 2658 3658 659 1659 2659 3659 660 1660 2660 3660 661 1661 2661 3661 662 1662 2662 3662 663 1663 2663 3663 664 1664 2664 3664 665 1665 2665 3665 666 1666 2666 3666 667 1667 2667 3667 668 1668 2668 3668 669 1669 2669 3669 670 1670 2670 3670 671 1671 2671 3671 672 1672 2672 3672 673 1673 2673 3673 674 1674 2674 3674 675 1675 2675 3675 676 1676 2676 3676 677 1677 2677 3677 678 1678 2678 3678 679 1679 2679 3679 680 1680 2680 3680 681 1681 2681 3681 682 1682 2682 3682 683 1683 2683 3683 684 1684 2684 3684 685 1685 2685 3685 686 1686 2686 3686 687 1687 2687 3687 688 1688 2688 3688 689 1689 2689 3689 690 1690 2690 3690 691 1691 2691 3691 692 1692 2692 3692 693 1693 2693 3693 694 1694 2694 3694 695 1695 2695 3695 696 1696 2696 3696 697 1697 2697 3697 698 1698 2698 3698 699 1699 2699 3699 700 1700 2700 3700 701 1701 2701 3701 702 1702 2702 3702 703 1703 2703 3703 704 1704 2704 3704 705 1705 2705 3705 706 1706 2706 3706 707 1707 2707 3707 708 1708 2708 3708 709 1709 2709 3709 710 1710 2710 3710 711 1711 2711 3711 712 1712 2712 3712 713 1713 2713 3713 714 1714 2714 3714 715 1715 2715 3715 716 1716 2716 3716 717 1717 2717 3717 718 1718 2718 3718 719 1719 2719 3719 720 1720 2720 3720 721 1721 2721 3721 722 1722 2722 3722 723 1723 2723 3723 724 1724 2724 3724 725 1725 2725 3725 726 1726 2726 3726 727 1727 2727 3727 728 1728 2728 3728 729 1729 2729 3729 730 1730 2730 3730 731 1731 2731 3731 732 1732 2732 3732 733 1733 2733 3733 734 1734 2734 3734 735 1735 2735 3735 736 1736 2736 3736 737 1737 2737 3737 738 1738 2738 3738 739 1739 2739 3739 740 1740 2740 3740 741 1741 2741 3741 742 1742 2742 3742 743 1743 2743 3743 744 1744 2744 3744 745 1745 2745 3745 746 1746 2746 3746 747 1747 2747 3747 748 1748 2748 3748 749 1749 2749 3749 750 1750 2750 3750 751 1751 2751 3751 752 1752 2752 3752 753 1753 2753 3753 754 1754 2754 3754 755 1755 2755 3755 756 1756 2756 3756 757 1757 2757 3757 758 1758 2758 3758 759 1759 2759 3759 760 1760 2760 3760 761 1761 2761 3761 762 1762 2762 3762 763 1763 2763 3763 764 1764 2764 3764 765 1765 2765 3765 766 1766 2766 3766 767 1767 2767 3767 768 1768 2768 3768 769 1769 2769 3769 770 1770 2770 3770 771 1771 2771 3771 772 1772 2772 3772 773 1773 2773 3773 774 1774 2774 3774 775 1775 2775 3775 776 1776 2776 3776 777 1777 2777 3777 778 1778 2778 3778 779 1779 2779 3779 780 1780 2780 3780 781 1781 2781 3781 782 1782 2782 3782 783 1783 2783 3783 784 1784 2784 3784 785 1785 2785 3785 786 1786 2786 3786 787 1787 2787 3787 788 1788 2788 3788 789 1789 2789 3789 790 1790 2790 3790 791 1791 2791 3791 792 1792 2792 3792 793 1793 2793 3793 794 1794 2794 3794 795 1795 2795 3795 796 1796 2796 3796 797 1797 2797 3797 798 1798 2798 3798 799 1799 2799 3799 800 1800 2800 3800 801 1801 2801 3801 802 1802 2802 3802 803 1803 2803 3803 804 1804 2804 3804 805 1805 2805 3805 806 1806 2806 3806 807 1807 2807 3807 808 1808 2808 3808 809 1809 2809 3809 810 1810 2810 3810 811 1811 2811 3811 812 1812 2812 3812 813 1813 2813 3813 814 1814 2814 3814 815 1815 2815 3815 816 1816 2816 3816 817 1817 2817 3817 818 1818 2818 3818 819 1819 2819 3819 820 1820 2820 3820 821 1821 2821 3821 822 1822 2822 3822 823 1823 2823 3823 824 1824 2824 3824 825 1825 2825 3825 826 1826 2826 3826 827 1827 2827 3827 828 1828 2828 3828 829 1829 2829 3829 830 1830 2830 3830 831 1831 2831 3831 832 1832 2832 3832 833 1833 2833 3833 834 1834 2834 3834 835 1835 2835 3835 836 1836 2836 3836 837 1837 2837 3837 838 1838 2838 3838 839 1839 2839 3839 840 1840 2840 3840 841 1841 2841 3841 842 1842 2842 3842 843 1843 2843 3843 844 1844 2844 3844 845 1845 2845 3845 846 1846 2846 3846 847 1847 2847 3847 848 1848 2848 3848 849 1849 2849 3849 850 1850 2850 3850 851 1851 2851 3851 852 1852 2852 3852 853 1853 2853 3853 854 1854 2854 3854 855 1855 2855 3855 856 1856 2856 3856 857 1857 2857 3857 858 1858 2858 3858 859 1859 2859 3859 860 1860 2860 3860 861 1861 2861 3861 862 1862 2862 3862 863 1863 2863 3863 864 1864 2864 3864 865 1865 2865 3865 866 1866 2866 3866 867 1867 2867 3867 868 1868 2868 3868 869 1869 2869 3869 870 1870 2870 3870 871 1871 2871 3871 872 1872 2872 3872 873 1873 2873 3873 874 1874 2874 3874 875 1875 2875 3875 876 1876 2876 3876 877 1877 2877 3877 878 1878 2878 3878 879 1879 2879 3879 880 1880 2880 3880 881 1881 2881 3881 882 1882 2882 3882 883 1883 2883 3883 884 1884 2884 3884 885 1885 2885 3885 886 1886 2886 3886 887 1887 2887 3887 888 1888 2888 3888 889 1889 2889 3889 890 1890 2890 3890 891 1891 2891 3891 892 1892 2892 3892 893 1893 2893 3893 894 1894 2894 3894 895 1895 2895 3895 896 1896 2896 3896 897 1897 2897 3897 898 1898 2898 3898 899 1899 2899 3899 900 1900 2900 3900 901 1901 2901 3901 902 1902 2902 3902 903 1903 2903 3903 904 1904 2904 3904 905 1905 2905 3905 906 1906 2906 3906 907 1907 2907 3907 908 1908 2908 3908 909 1909 2909 3909 910 1910 2910 3910 911 1911 2911 3911 912 1912 2912 3912 913 1913 2913 3913 914 1914 2914 3914 915 1915 2915 3915 916 1916 2916 3916 917 1917 2917 3917 918 1918 2918 3918 919 1919 2919 3919 920 1920 2920 3920 921 1921 2921 3921 922 1922 2922 3922 923 1923 2923 3923 924 1924 2924 3924 925 1925 2925 3925 926 1926 2926 3926 927 1927 2927 3927 928 1928 2928 3928 929 1929 2929 3929 930 1930 2930 3930 931 1931 2931 3931 932 1932 2932 3932 933 1933 2933 3933 934 1934 2934 3934 935 1935 2935 3935 936 1936 2936 3936 937 1937 2937 3937 938 1938 2938 3938 939 1939 2939 3939 940 1940 2940 3940 941 1941 2941 3941 942 1942 2942 3942 943 1943 2943 3943 944 1944 2944 3944 945 1945 2945 3945 946 1946 2946 3946 947 1947 2947 3947 948 1948 2948 3948 949 1949 2949 3949 950 1950 2950 3950 951 1951 2951 3951 952 1952 2952 3952 953 1953 2953 3953 954 1954 2954 3954 955 1955 2955 3955 956 1956 2956 3956 957 1957 2957 3957 958 1958 2958 3958 959 1959 2959 3959 960 1960 2960 3960 961 1961 2961 3961 962 1962 2962 3962 963 1963 2963 3963 964 1964 2964 3964 965 1965 2965 3965 966 1966 2966 3966 967 1967 2967 3967 968 1968 2968 3968 969 1969 2969 3969 970 1970 2970 3970 971 1971 2971 3971 972 1972 2972 3972 973 1973 2973 3973 974 1974 2974 3974 975 1975 2975 3975 976 1976 2976 3976 977 1977 2977 3977 978 1978 2978 3978 979 1979 2979 3979 980 1980 2980 3980 981 1981 2981 3981 982 1982 2982 3982 983 1983 2983 3983 984 1984 2984 3984 985 1985 2985 3985 986 1986 2986 3986 987 1987 2987 3987 988 1988 2988 3988 989 1989 2989 3989 990 1990 2990 3990 991 1991 2991 3991 992 1992 2992 3992 993 1993 2993 3993 994 1994 2994 3994 995 1995 2995 3995 996 1996 2996 3996 997 1997 2997 3997 998 1998 2998 3998 999 1999 2999 3999 1000 2000 3000 4000 1001 2001 3001 4001 1002 2002 3002 4002

In some embodiments, the sequences that specifically bind to almond comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 4003 to 5002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to almond may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5003 to 6002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to almond may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 6003 to 7002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to almond may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 7003 to 8002. In one embodiment, the aptamer of the present disclosure that specifically binds to almond may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 4003 to 8002 listed in Table 3, or variant thereof.

TABLE 3 Aptamer sequences against almond Aptamer Sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 4003 5003 6003 7003 4004 5004 6004 7004 4005 5005 6005 7005 4006 5006 6006 7006 4007 5007 6007 7007 4008 5008 6008 7008 4009 5009 6009 7009 4010 5010 6010 7010 4011 5011 6011 7011 4012 5012 6012 7012 4013 5013 6013 7013 4014 5014 6014 7014 4015 5015 6015 7015 4016 5016 6016 7016 4017 5017 6017 7017 4018 5018 6018 7018 4019 5019 6019 7019 4020 5020 6020 7020 4021 5021 6021 7021 4022 5022 6022 7022 4023 5023 6023 7023 4024 5024 6024 7024 4025 5025 6025 7025 4026 5026 6026 7026 4027 5027 6027 7027 4028 5028 6028 7028 4029 5029 6029 7029 4030 5030 6030 7030 4031 5031 6031 7031 4032 5032 6032 7032 4033 5033 6033 7033 4034 5034 6034 7034 4035 5035 6035 7035 4036 5036 6036 7036 4037 5037 6037 7037 4038 5038 6038 7038 4039 5039 6039 7039 4040 5040 6040 7040 4041 5041 6041 7041 4042 5042 6042 7042 4043 5043 6043 7043 4044 5044 6044 7044 4045 5045 6045 7045 4046 5046 6046 7046 4047 5047 6047 7047 4048 5048 6048 7048 4049 5049 6049 7049 4050 5050 6050 7050 4051 5051 6051 7051 4052 5052 6052 7052 4053 5053 6053 7053 4054 5054 6054 7054 4055 5055 6055 7055 4056 5056 6056 7056 4057 5057 6057 7057 4058 5058 6058 7058 4059 5059 6059 7059 4060 5060 6060 7060 4061 5061 6061 7061 4062 5062 6062 7062 4063 5063 6063 7063 4064 5064 6064 7064 4065 5065 6065 7065 4066 5066 6066 7066 4067 5067 6067 7067 4068 5068 6068 7068 4069 5069 6069 7069 4070 5070 6070 7070 4071 5071 6071 7071 4072 5072 6072 7072 4073 5073 6073 7073 4074 5074 6074 7074 4075 5075 6075 7075 4076 5076 6076 7076 4077 5077 6077 7077 4078 5078 6078 7078 4079 5079 6079 7079 4080 5080 6080 7080 4081 5081 6081 7081 4082 5082 6082 7082 4083 5083 6083 7083 4084 5084 6084 7084 4085 5085 6085 7085 4086 5086 6086 7086 4087 5087 6087 7087 4088 5088 6088 7088 4089 5089 6089 7089 4090 5090 6090 7090 4091 5091 6091 7091 4092 5092 6092 7092 4093 5093 6093 7093 4094 5094 6094 7094 4095 5095 6095 7095 4096 5096 6096 7096 4097 5097 6097 7097 4098 5098 6098 7098 4099 5099 6099 7099 4100 5100 6100 7100 4101 5101 6101 7101 4102 5102 6102 7102 4103 5103 6103 7103 4104 5104 6104 7104 4105 5105 6105 7105 4106 5106 6106 7106 4107 5107 6107 7107 4108 5108 6108 7108 4109 5109 6109 7109 4110 5110 6110 7110 4111 5111 6111 7111 4112 5112 6112 7112 4113 5113 6113 7113 4114 5114 6114 7114 4115 5115 6115 7115 4116 5116 6116 7116 4117 5117 6117 7117 4118 5118 6118 7118 4119 5119 6119 7119 4120 5120 6120 7120 4121 5121 6121 7121 4122 5122 6122 7122 4123 5123 6123 7123 4124 5124 6124 7124 4125 5125 6125 7125 4126 5126 6126 7126 4127 5127 6127 7127 4128 5128 6128 7128 4129 5129 6129 7129 4130 5130 6130 7130 4131 5131 6131 7131 4132 5132 6132 7132 4133 5133 6133 7133 4134 5134 6134 7134 4135 5135 6135 7135 4136 5136 6136 7136 4137 5137 6137 7137 4138 5138 6138 7138 4139 5139 6139 7139 4140 5140 6140 7140 4141 5141 6141 7141 4142 5142 6142 7142 4143 5143 6143 7143 4144 5144 6144 7144 4145 5145 6145 7145 4146 5146 6146 7146 4147 5147 6147 7147 4148 5148 6148 7148 4149 5149 6149 7149 4150 5150 6150 7150 4151 5151 6151 7151 4152 5152 6152 7152 4153 5153 6153 7153 4154 5154 6154 7154 4155 5155 6155 7155 4156 5156 6156 7156 4157 5157 6157 7157 4158 5158 6158 7158 4159 5159 6159 7159 4160 5160 6160 7160 4161 5161 6161 7161 4162 5162 6162 7162 4163 5163 6163 7163 4164 5164 6164 7164 4165 5165 6165 7165 4166 5166 6166 7166 4167 5167 6167 7167 4168 5168 6168 7168 4169 5169 6169 7169 4170 5170 6170 7170 4171 5171 6171 7171 4172 5172 6172 7172 4173 5173 6173 7173 4174 5174 6174 7174 4175 5175 6175 7175 4176 5176 6176 7176 4177 5177 6177 7177 4178 5178 6178 7178 4179 5179 6179 7179 4180 5180 6180 7180 4181 5181 6181 7181 4182 5182 6182 7182 4183 5183 6183 7183 4184 5184 6184 7184 4185 5185 6185 7185 4186 5186 6186 7186 4187 5187 6187 7187 4188 5188 6188 7188 4189 5189 6189 7189 4190 5190 6190 7190 4191 5191 6191 7191 4192 5192 6192 7192 4193 5193 6193 7193 4194 5194 6194 7194 4195 5195 6195 7195 4196 5196 6196 7196 4197 5197 6197 7197 4198 5198 6198 7198 4199 5199 6199 7199 4200 5200 6200 7200 4201 5201 6201 7201 4202 5202 6202 7202 4203 5203 6203 7203 4204 5204 6204 7204 4205 5205 6205 7205 4206 5206 6206 7206 4207 5207 6207 7207 4208 5208 6208 7208 4209 5209 6209 7209 4210 5210 6210 7210 4211 5211 6211 7211 4212 5212 6212 7212 4213 5213 6213 7213 4214 5214 6214 7214 4215 5215 6215 7215 4216 5216 6216 7216 4217 5217 6217 7217 4218 5218 6218 7218 4219 5219 6219 7219 4220 5220 6220 7220 4221 5221 6221 7221 4222 5222 6222 7222 4223 5223 6223 7223 4224 5224 6224 7224 4225 5225 6225 7225 4226 5226 6226 7226 4227 5227 6227 7227 4228 5228 6228 7228 4229 5229 6229 7229 4230 5230 6230 7230 4231 5231 6231 7231 4232 5232 6232 7232 4233 5233 6233 7233 4234 5234 6234 7234 4235 5235 6235 7235 4236 5236 6236 7236 4237 5237 6237 7237 4238 5238 6238 7238 4239 5239 6239 7239 4240 5240 6240 7240 4241 5241 6241 7241 4242 5242 6242 7242 4243 5243 6243 7243 4244 5244 6244 7244 4245 5245 6245 7245 4246 5246 6246 7246 4247 5247 6247 7247 4248 5248 6248 7248 4249 5249 6249 7249 4250 5250 6250 7250 4251 5251 6251 7251 4252 5252 6252 7252 4253 5253 6253 7253 4254 5254 6254 7254 4255 5255 6255 7255 4256 5256 6256 7256 4257 5257 6257 7257 4258 5258 6258 7258 4259 5259 6259 7259 4260 5260 6260 7260 4261 5261 6261 7261 4262 5262 6262 7262 4263 5263 6263 7263 4264 5264 6264 7264 4265 5265 6265 7265 4266 5266 6266 7266 4267 5267 6267 7267 4268 5268 6268 7268 4269 5269 6269 7269 4270 5270 6270 7270 4271 5271 6271 7271 4272 5272 6272 7272 4273 5273 6273 7273 4274 5274 6274 7274 4275 5275 6275 7275 4276 5276 6276 7276 4277 5277 6277 7277 4278 5278 6278 7278 4279 5279 6279 7279 4280 5280 6280 7280 4281 5281 6281 7281 4282 5282 6282 7282 4283 5283 6283 7283 4284 5284 6284 7284 4285 5285 6285 7285 4286 5286 6286 7286 4287 5287 6287 7287 4288 5288 6288 7288 4289 5289 6289 7289 4290 5290 6290 7290 4291 5291 6291 7291 4292 5292 6292 7292 4293 5293 6293 7293 4294 5294 6294 7294 4295 5295 6295 7295 4296 5296 6296 7296 4297 5297 6297 7297 4298 5298 6298 7298 4299 5299 6299 7299 4300 5300 6300 7300 4301 5301 6301 7301 4302 5302 6302 7302 4303 5303 6303 7303 4304 5304 6304 7304 4305 5305 6305 7305 4306 5306 6306 7306 4307 5307 6307 7307 4308 5308 6308 7308 4309 5309 6309 7309 4310 5310 6310 7310 4311 5311 6311 7311 4312 5312 6312 7312 4313 5313 6313 7313 4314 5314 6314 7314 4315 5315 6315 7315 4316 5316 6316 7316 4317 5317 6317 7317 4318 5318 6318 7318 4319 5319 6319 7319 4320 5320 6320 7320 4321 5321 6321 7321 4322 5322 6322 7322 4323 5323 6323 7323 4324 5324 6324 7324 4325 5325 6325 7325 4326 5326 6326 7326 4327 5327 6327 7327 4328 5328 6328 7328 4329 5329 6329 7329 4330 5330 6330 7330 4331 5331 6331 7331 4332 5332 6332 7332 4333 5333 6333 7333 4334 5334 6334 7334 4335 5335 6335 7335 4336 5336 6336 7336 4337 5337 6337 7337 4338 5338 6338 7338 4339 5339 6339 7339 4340 5340 6340 7340 4341 5341 6341 7341 4342 5342 6342 7342 4343 5343 6343 7343 4344 5344 6344 7344 4345 5345 6345 7345 4346 5346 6346 7346 4347 5347 6347 7347 4348 5348 6348 7348 4349 5349 6349 7349 4350 5350 6350 7350 4351 5351 6351 7351 4352 5352 6352 7352 4353 5353 6353 7353 4354 5354 6354 7354 4355 5355 6355 7355 4356 5356 6356 7356 4357 5357 6357 7357 4358 5358 6358 7358 4359 5359 6359 7359 4360 5360 6360 7360 4361 5361 6361 7361 4362 5362 6362 7362 4363 5363 6363 7363 4364 5364 6364 7364 4365 5365 6365 7365 4366 5366 6366 7366 4367 5367 6367 7367 4368 5368 6368 7368 4369 5369 6369 7369 4370 5370 6370 7370 4371 5371 6371 7371 4372 5372 6372 7372 4373 5373 6373 7373 4374 5374 6374 7374 4375 5375 6375 7375 4376 5376 6376 7376 4377 5377 6377 7377 4378 5378 6378 7378 4379 5379 6379 7379 4380 5380 6380 7380 4381 5381 6381 7381 4382 5382 6382 7382 4383 5383 6383 7383 4384 5384 6384 7384 4385 5385 6385 7385 4386 5386 6386 7386 4387 5387 6387 7387 4388 5388 6388 7388 4389 5389 6389 7389 4390 5390 6390 7390 4391 5391 6391 7391 4392 5392 6392 7392 4393 5393 6393 7393 4394 5394 6394 7394 4395 5395 6395 7395 4396 5396 6396 7396 4397 5397 6397 7397 4398 5398 6398 7398 4399 5399 6399 7399 4400 5400 6400 7400 4401 5401 6401 7401 4402 5402 6402 7402 4403 5403 6403 7403 4404 5404 6404 7404 4405 5405 6405 7405 4406 5406 6406 7406 4407 5407 6407 7407 4408 5408 6408 7408 4409 5409 6409 7409 4410 5410 6410 7410 4411 5411 6411 7411 4412 5412 6412 7412 4413 5413 6413 7413 4414 5414 6414 7414 4415 5415 6415 7415 4416 5416 6416 7416 4417 5417 6417 7417 4418 5418 6418 7418 4419 5419 6419 7419 4420 5420 6420 7420 4421 5421 6421 7421 4422 5422 6422 7422 4423 5423 6423 7423 4424 5424 6424 7424 4425 5425 6425 7425 4426 5426 6426 7426 4427 5427 6427 7427 4428 5428 6428 7428 4429 5429 6429 7429 4430 5430 6430 7430 4431 5431 6431 7431 4432 5432 6432 7432 4433 5433 6433 7433 4434 5434 6434 7434 4435 5435 6435 7435 4436 5436 6436 7436 4437 5437 6437 7437 4438 5438 6438 7438 4439 5439 6439 7439 4440 5440 6440 7440 4441 5441 6441 7441 4442 5442 6442 7442 4443 5443 6443 7443 4444 5444 6444 7444 4445 5445 6445 7445 4446 5446 6446 7446 4447 5447 6447 7447 4448 5448 6448 7448 4449 5449 6449 7449 4450 5450 6450 7450 4451 5451 6451 7451 4452 5452 6452 7452 4453 5453 6453 7453 4454 5454 6454 7454 4455 5455 6455 7455 4456 5456 6456 7456 4457 5457 6457 7457 4458 5458 6458 7458 4459 5459 6459 7459 4460 5460 6460 7460 4461 5461 6461 7461 4462 5462 6462 7462 4463 5463 6463 7463 4464 5464 6464 7464 4465 5465 6465 7465 4466 5466 6466 7466 4467 5467 6467 7467 4468 5468 6468 7468 4469 5469 6469 7469 4470 5470 6470 7470 4471 5471 6471 7471 4472 5472 6472 7472 4473 5473 6473 7473 4474 5474 6474 7474 4475 5475 6475 7475 4476 5476 6476 7476 4477 5477 6477 7477 4478 5478 6478 7478 4479 5479 6479 7479 4480 5480 6480 7480 4481 5481 6481 7481 4482 5482 6482 7482 4483 5483 6483 7483 4484 5484 6484 7484 4485 5485 6485 7485 4486 5486 6486 7486 4487 5487 6487 7487 4488 5488 6488 7488 4489 5489 6489 7489 4490 5490 6490 7490 4491 5491 6491 7491 4492 5492 6492 7492 4493 5493 6493 7493 4494 5494 6494 7494 4495 5495 6495 7495 4496 5496 6496 7496 4497 5497 6497 7497 4498 5498 6498 7498 4499 5499 6499 7499 4500 5500 6500 7500 4501 5501 6501 7501 4502 5502 6502 7502 4503 5503 6503 7503 4504 5504 6504 7504 4505 5505 6505 7505 4506 5506 6506 7506 4507 5507 6507 7507 4508 5508 6508 7508 4509 5509 6509 7509 4510 5510 6510 7510 4511 5511 6511 7511 4512 5512 6512 7512 4513 5513 6513 7513 4514 5514 6514 7514 4515 5515 6515 7515 4516 5516 6516 7516 4517 5517 6517 7517 4518 5518 6518 7518 4519 5519 6519 7519 4520 5520 6520 7520 4521 5521 6521 7521 4522 5522 6522 7522 4523 5523 6523 7523 4524 5524 6524 7524 4525 5525 6525 7525 4526 5526 6526 7526 4527 5527 6527 7527 4528 5528 6528 7528 4529 5529 6529 7529 4530 5530 6530 7530 4531 5531 6531 7531 4532 5532 6532 7532 4533 5533 6533 7533 4534 5534 6534 7534 4535 5535 6535 7535 4536 5536 6536 7536 4537 5537 6537 7537 4538 5538 6538 7538 4539 5539 6539 7539 4540 5540 6540 7540 4541 5541 6541 7541 4542 5542 6542 7542 4543 5543 6543 7543 4544 5544 6544 7544 4545 5545 6545 7545 4546 5546 6546 7546 4547 5547 6547 7547 4548 5548 6548 7548 4549 5549 6549 7549 4550 5550 6550 7550 4551 5551 6551 7551 4552 5552 6552 7552 4553 5553 6553 7553 4554 5554 6554 7554 4555 5555 6555 7555 4556 5556 6556 7556 4557 5557 6557 7557 4558 5558 6558 7558 4559 5559 6559 7559 4560 5560 6560 7560 4561 5561 6561 7561 4562 5562 6562 7562 4563 5563 6563 7563 4564 5564 6564 7564 4565 5565 6565 7565 4566 5566 6566 7566 4567 5567 6567 7567 4568 5568 6568 7568 4569 5569 6569 7569 4570 5570 6570 7570 4571 5571 6571 7571 4572 5572 6572 7572 4573 5573 6573 7573 4574 5574 6574 7574 4575 5575 6575 7575 4576 5576 6576 7576 4577 5577 6577 7577 4578 5578 6578 7578 4579 5579 6579 7579 4580 5580 6580 7580 4581 5581 6581 7581 4582 5582 6582 7582 4583 5583 6583 7583 4584 5584 6584 7584 4585 5585 6585 7585 4586 5586 6586 7586 4587 5587 6587 7587 4588 5588 6588 7588 4589 5589 6589 7589 4590 5590 6590 7590 4591 5591 6591 7591 4592 5592 6592 7592 4593 5593 6593 7593 4594 5594 6594 7594 4595 5595 6595 7595 4596 5596 6596 7596 4597 5597 6597 7597 4598 5598 6598 7598 4599 5599 6599 7599 4600 5600 6600 7600 4601 5601 6601 7601 4602 5602 6602 7602 4603 5603 6603 7603 4604 5604 6604 7604 4605 5605 6605 7605 4606 5606 6606 7606 4607 5607 6607 7607 4608 5608 6608 7608 4609 5609 6609 7609 4610 5610 6610 7610 4611 5611 6611 7611 4612 5612 6612 7612 4613 5613 6613 7613 4614 5614 6614 7614 4615 5615 6615 7615 4616 5616 6616 7616 4617 5617 6617 7617 4618 5618 6618 7618 4619 5619 6619 7619 4620 5620 6620 7620 4621 5621 6621 7621 4622 5622 6622 7622 4623 5623 6623 7623 4624 5624 6624 7624 4625 5625 6625 7625 4626 5626 6626 7626 4627 5627 6627 7627 4628 5628 6628 7628 4629 5629 6629 7629 4630 5630 6630 7630 4631 5631 6631 7631 4632 5632 6632 7632 4633 5633 6633 7633 4634 5634 6634 7634 4635 5635 6635 7635 4636 5636 6636 7636 4637 5637 6637 7637 4638 5638 6638 7638 4639 5639 6639 7639 4640 5640 6640 7640 4641 5641 6641 7641 4642 5642 6642 7642 4643 5643 6643 7643 4644 5644 6644 7644 4645 5645 6645 7645 4646 5646 6646 7646 4647 5647 6647 7647 4648 5648 6648 7648 4649 5649 6649 7649 4650 5650 6650 7650 4651 5651 6651 7651 4652 5652 6652 7652 4653 5653 6653 7653 4654 5654 6654 7654 4655 5655 6655 7655 4656 5656 6656 7656 4657 5657 6657 7657 4658 5658 6658 7658 4659 5659 6659 7659 4660 5660 6660 7660 4661 5661 6661 7661 4662 5662 6662 7662 4663 5663 6663 7663 4664 5664 6664 7664 4665 5665 6665 7665 4666 5666 6666 7666 4667 5667 6667 7667 4668 5668 6668 7668 4669 5669 6669 7669 4670 5670 6670 7670 4671 5671 6671 7671 4672 5672 6672 7672 4673 5673 6673 7673 4674 5674 6674 7674 4675 5675 6675 7675 4676 5676 6676 7676 4677 5677 6677 7677 4678 5678 6678 7678 4679 5679 6679 7679 4680 5680 6680 7680 4681 5681 6681 7681 4682 5682 6682 7682 4683 5683 6683 7683 4684 5684 6684 7684 4685 5685 6685 7685 4686 5686 6686 7686 4687 5687 6687 7687 4688 5688 6688 7688 4689 5689 6689 7689 4690 5690 6690 7690 4691 5691 6691 7691 4692 5692 6692 7692 4693 5693 6693 7693 4694 5694 6694 7694 4695 5695 6695 7695 4696 5696 6696 7696 4697 5697 6697 7697 4698 5698 6698 7698 4699 5699 6699 7699 4700 5700 6700 7700 4701 5701 6701 7701 4702 5702 6702 7702 4703 5703 6703 7703 4704 5704 6704 7704 4705 5705 6705 7705 4706 5706 6706 7706 4707 5707 6707 7707 4708 5708 6708 7708 4709 5709 6709 7709 4710 5710 6710 7710 4711 5711 6711 7711 4712 5712 6712 7712 4713 5713 6713 7713 4714 5714 6714 7714 4715 5715 6715 7715 4716 5716 6716 7716 4717 5717 6717 7717 4718 5718 6718 7718 4719 5719 6719 7719 4720 5720 6720 7720 4721 5721 6721 7721 4722 5722 6722 7722 4723 5723 6723 7723 4724 5724 6724 7724 4725 5725 6725 7725 4726 5726 6726 7726 4727 5727 6727 7727 4728 5728 6728 7728 4729 5729 6729 7729 4730 5730 6730 7730 4731 5731 6731 7731 4732 5732 6732 7732 4733 5733 6733 7733 4734 5734 6734 7734 4735 5735 6735 7735 4736 5736 6736 7736 4737 5737 6737 7737 4738 5738 6738 7738 4739 5739 6739 7739 4740 5740 6740 7740 4741 5741 6741 7741 4742 5742 6742 7742 4743 5743 6743 7743 4744 5744 6744 7744 4745 5745 6745 7745 4746 5746 6746 7746 4747 5747 6747 7747 4748 5748 6748 7748 4749 5749 6749 7749 4750 5750 6750 7750 4751 5751 6751 7751 4752 5752 6752 7752 4753 5753 6753 7753 4754 5754 6754 7754 4755 5755 6755 7755 4756 5756 6756 7756 4757 5757 6757 7757 4758 5758 6758 7758 4759 5759 6759 7759 4760 5760 6760 7760 4761 5761 6761 7761 4762 5762 6762 7762 4763 5763 6763 7763 4764 5764 6764 7764 4765 5765 6765 7765 4766 5766 6766 7766 4767 5767 6767 7767 4768 5768 6768 7768 4769 5769 6769 7769 4770 5770 6770 7770 4771 5771 6771 7771 4772 5772 6772 7772 4773 5773 6773 7773 4774 5774 6774 7774 4775 5775 6775 7775 4776 5776 6776 7776 4777 5777 6777 7777 4778 5778 6778 7778 4779 5779 6779 7779 4780 5780 6780 7780 4781 5781 6781 7781 4782 5782 6782 7782 4783 5783 6783 7783 4784 5784 6784 7784 4785 5785 6785 7785 4786 5786 6786 7786 4787 5787 6787 7787 4788 5788 6788 7788 4789 5789 6789 7789 4790 5790 6790 7790 4791 5791 6791 7791 4792 5792 6792 7792 4793 5793 6793 7793 4794 5794 6794 7794 4795 5795 6795 7795 4796 5796 6796 7796 4797 5797 6797 7797 4798 5798 6798 7798 4799 5799 6799 7799 4800 5800 6800 7800 4801 5801 6801 7801 4802 5802 6802 7802 4803 5803 6803 7803 4804 5804 6804 7804 4805 5805 6805 7805 4806 5806 6806 7806 4807 5807 6807 7807 4808 5808 6808 7808 4809 5809 6809 7809 4810 5810 6810 7810 4811 5811 6811 7811 4812 5812 6812 7812 4813 5813 6813 7813 4814 5814 6814 7814 4815 5815 6815 7815 4816 5816 6816 7816 4817 5817 6817 7817 4818 5818 6818 7818 4819 5819 6819 7819 4820 5820 6820 7820 4821 5821 6821 7821 4822 5822 6822 7822 4823 5823 6823 7823 4824 5824 6824 7824 4825 5825 6825 7825 4826 5826 6826 7826 4827 5827 6827 7827 4828 5828 6828 7828 4829 5829 6829 7829 4830 5830 6830 7830 4831 5831 6831 7831 4832 5832 6832 7832 4833 5833 6833 7833 4834 5834 6834 7834 4835 5835 6835 7835 4836 5836 6836 7836 4837 5837 6837 7837 4838 5838 6838 7838 4839 5839 6839 7839 4840 5840 6840 7840 4841 5841 6841 7841 4842 5842 6842 7842 4843 5843 6843 7843 4844 5844 6844 7844 4845 5845 6845 7845 4846 5846 6846 7846 4847 5847 6847 7847 4848 5848 6848 7848 4849 5849 6849 7849 4850 5850 6850 7850 4851 5851 6851 7851 4852 5852 6852 7852 4853 5853 6853 7853 4854 5854 6854 7854 4855 5855 6855 7855 4856 5856 6856 7856 4857 5857 6857 7857 4858 5858 6858 7858 4859 5859 6859 7859 4860 5860 6860 7860 4861 5861 6861 7861 4862 5862 6862 7862 4863 5863 6863 7863 4864 5864 6864 7864 4865 5865 6865 7865 4866 5866 6866 7866 4867 5867 6867 7867 4868 5868 6868 7868 4869 5869 6869 7869 4870 5870 6870 7870 4871 5871 6871 7871 4872 5872 6872 7872 4873 5873 6873 7873 4874 5874 6874 7874 4875 5875 6875 7875 4876 5876 6876 7876 4877 5877 6877 7877 4878 5878 6878 7878 4879 5879 6879 7879 4880 5880 6880 7880 4881 5881 6881 7881 4882 5882 6882 7882 4883 5883 6883 7883 4884 5884 6884 7884 4885 5885 6885 7885 4886 5886 6886 7886 4887 5887 6887 7887 4888 5888 6888 7888 4889 5889 6889 7889 4890 5890 6890 7890 4891 5891 6891 7891 4892 5892 6892 7892 4893 5893 6893 7893 4894 5894 6894 7894 4895 5895 6895 7895 4896 5896 6896 7896 4897 5897 6897 7897 4898 5898 6898 7898 4899 5899 6899 7899 4900 5900 6900 7900 4901 5901 6901 7901 4902 5902 6902 7902 4903 5903 6903 7903 4904 5904 6904 7904 4905 5905 6905 7905 4906 5906 6906 7906 4907 5907 6907 7907 4908 5908 6908 7908 4909 5909 6909 7909 4910 5910 6910 7910 4911 5911 6911 7911 4912 5912 6912 7912 4913 5913 6913 7913 4914 5914 6914 7914 4915 5915 6915 7915 4916 5916 6916 7916 4917 5917 6917 7917 4918 5918 6918 7918 4919 5919 6919 7919 4920 5920 6920 7920 4921 5921 6921 7921 4922 5922 6922 7922 4923 5923 6923 7923 4924 5924 6924 7924 4925 5925 6925 7925 4926 5926 6926 7926 4927 5927 6927 7927 4928 5928 6928 7928 4929 5929 6929 7929 4930 5930 6930 7930 4931 5931 6931 7931 4932 5932 6932 7932 4933 5933 6933 7933 4934 5934 6934 7934 4935 5935 6935 7935 4936 5936 6936 7936 4937 5937 6937 7937 4938 5938 6938 7938 4939 5939 6939 7939 4940 5940 6940 7940 4941 5941 6941 7941 4942 5942 6942 7942 4943 5943 6943 7943 4944 5944 6944 7944 4945 5945 6945 7945 4946 5946 6946 7946 4947 5947 6947 7947 4948 5948 6948 7948 4949 5949 6949 7949 4950 5950 6950 7950 4951 5951 6951 7951 4952 5952 6952 7952 4953 5953 6953 7953 4954 5954 6954 7954 4955 5955 6955 7955 4956 5956 6956 7956 4957 5957 6957 7957 4958 5958 6958 7958 4959 5959 6959 7959 4960 5960 6960 7960 4961 5961 6961 7961 4962 5962 6962 7962 4963 5963 6963 7963 4964 5964 6964 7964 4965 5965 6965 7965 4966 5966 6966 7966 4967 5967 6967 7967 4968 5968 6968 7968 4969 5969 6969 7969 4970 5970 6970 7970 4971 5971 6971 7971 4972 5972 6972 7972 4973 5973 6973 7973 4974 5974 6974 7974 4975 5975 6975 7975 4976 5976 6976 7976 4977 5977 6977 7977 4978 5978 6978 7978 4979 5979 6979 7979 4980 5980 6980 7980 4981 5981 6981 7981 4982 5982 6982 7982 4983 5983 6983 7983 4984 5984 6984 7984 4985 5985 6985 7985 4986 5986 6986 7986 4987 5987 6987 7987 4988 5988 6988 7988 4989 5989 6989 7989 4990 5990 6990 7990 4991 5991 6991 7991 4992 5992 6992 7992 4993 5993 6993 7993 4994 5994 6994 7994 4995 5995 6995 7995 4996 5996 6996 7996 4997 5997 6997 7997 4998 5998 6998 7998 4999 5999 6999 7999 5000 6000 7000 8000 5001 6001 7001 8001 5002 6002 7002 8002

In some embodiments, the sequences that specifically bind to brazil nut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 8003 to 9002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to brazil nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9003 to 10002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to brazil nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 10003 to 11002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to brazil nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 11003 to 12002. In one embodiment, the aptamer of the present disclosure that specifically binds to brazil nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 8003 to 12002 listed in Table 4, or variant thereof.

TABLE 4 Aptamer sequences against brazil nut Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 8003 9003 10003 11003 8004 9004 10004 11004 8005 9005 10005 11005 8006 9006 10006 11006 8007 9007 10007 11007 8008 9008 10008 11008 8009 9009 10009 11009 8010 9010 10010 11010 8011 9011 10011 11011 8012 9012 10012 11012 8013 9013 10013 11013 8014 9014 10014 11014 8015 9015 10015 11015 8016 9016 10016 11016 8017 9017 10017 11017 8018 9018 10018 11018 8019 9019 10019 11019 8020 9020 10020 11020 8021 9021 10021 11021 8022 9022 10022 11022 8023 9023 10023 11023 8024 9024 10024 11024 8025 9025 10025 11025 8026 9026 10026 11026 8027 9027 10027 11027 8028 9028 10028 11028 8029 9029 10029 11029 8030 9030 10030 11030 8031 9031 10031 11031 8032 9032 10032 11032 8033 9033 10033 11033 8034 9034 10034 11034 8035 9035 10035 11035 8036 9036 10036 11036 8037 9037 10037 11037 8038 9038 10038 11038 8039 9039 10039 11039 8040 9040 10040 11040 8041 9041 10041 11041 8042 9042 10042 11042 8043 9043 10043 11043 8044 9044 10044 11044 8045 9045 10045 11045 8046 9046 10046 11046 8047 9047 10047 11047 8048 9048 10048 11048 8049 9049 10049 11049 8050 9050 10050 11050 8051 9051 10051 11051 8052 9052 10052 11052 8053 9053 10053 11053 8054 9054 10054 11054 8055 9055 10055 11055 8056 9056 10056 11056 8057 9057 10057 11057 8058 9058 10058 11058 8059 9059 10059 11059 8060 9060 10060 11060 8061 9061 10061 11061 8062 9062 10062 11062 8063 9063 10063 11063 8064 9064 10064 11064 8065 9065 10065 11065 8066 9066 10066 11066 8067 9067 10067 11067 8068 9068 10068 11068 8069 9069 10069 11069 8070 9070 10070 11070 8071 9071 10071 11071 8072 9072 10072 11072 8073 9073 10073 11073 8074 9074 10074 11074 8075 9075 10075 11075 8076 9076 10076 11076 8077 9077 10077 11077 8078 9078 10078 11078 8079 9079 10079 11079 8080 9080 10080 11080 8081 9081 10081 11081 8082 9082 10082 11082 8083 9083 10083 11083 8084 9084 10084 11084 8085 9085 10085 11085 8086 9086 10086 11086 8087 9087 10087 11087 8088 9088 10088 11088 8089 9089 10089 11089 8090 9090 10090 11090 8091 9091 10091 11091 8092 9092 10092 11092 8093 9093 10093 11093 8094 9094 10094 11094 8095 9095 10095 11095 8096 9096 10096 11096 8097 9097 10097 11097 8098 9098 10098 11098 8099 9099 10099 11099 8100 9100 10100 11100 8101 9101 10101 11101 8102 9102 10102 11102 8103 9103 10103 11103 8104 9104 10104 11104 8105 9105 10105 11105 8106 9106 10106 11106 8107 9107 10107 11107 8108 9108 10108 11108 8109 9109 10109 11109 8110 9110 10110 11110 8111 9111 10111 11111 8112 9112 10112 11112 8113 9113 10113 11113 8114 9114 10114 11114 8115 9115 10115 11115 8116 9116 10116 11116 8117 9117 10117 11117 8118 9118 10118 11118 8119 9119 10119 11119 8120 9120 10120 11120 8121 9121 10121 11121 8122 9122 10122 11122 8123 9123 10123 11123 8124 9124 10124 11124 8125 9125 10125 11125 8126 9126 10126 11126 8127 9127 10127 11127 8128 9128 10128 11128 8129 9129 10129 11129 8130 9130 10130 11130 8131 9131 10131 11131 8132 9132 10132 11132 8133 9133 10133 11133 8134 9134 10134 11134 8135 9135 10135 11135 8136 9136 10136 11136 8137 9137 10137 11137 8138 9138 10138 11138 8139 9139 10139 11139 8140 9140 10140 11140 8141 9141 10141 11141 8142 9142 10142 11142 8143 9143 10143 11143 8144 9144 10144 11144 8145 9145 10145 11145 8146 9146 10146 11146 8147 9147 10147 11147 8148 9148 10148 11148 8149 9149 10149 11149 8150 9150 10150 11150 8151 9151 10151 11151 8152 9152 10152 11152 8153 9153 10153 11153 8154 9154 10154 11154 8155 9155 10155 11155 8156 9156 10156 11156 8157 9157 10157 11157 8158 9158 10158 11158 8159 9159 10159 11159 8160 9160 10160 11160 8161 9161 10161 11161 8162 9162 10162 11162 8163 9163 10163 11163 8164 9164 10164 11164 8165 9165 10165 11165 8166 9166 10166 11166 8167 9167 10167 11167 8168 9168 10168 11168 8169 9169 10169 11169 8170 9170 10170 11170 8171 9171 10171 11171 8172 9172 10172 11172 8173 9173 10173 11173 8174 9174 10174 11174 8175 9175 10175 11175 8176 9176 10176 11176 8177 9177 10177 11177 8178 9178 10178 11178 8179 9179 10179 11179 8180 9180 10180 11180 8181 9181 10181 11181 8182 9182 10182 11182 8183 9183 10183 11183 8184 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11229 8230 9230 10230 11230 8231 9231 10231 11231 8232 9232 10232 11232 8233 9233 10233 11233 8234 9234 10234 11234 8235 9235 10235 11235 8236 9236 10236 11236 8237 9237 10237 11237 8238 9238 10238 11238 8239 9239 10239 11239 8240 9240 10240 11240 8241 9241 10241 11241 8242 9242 10242 11242 8243 9243 10243 11243 8244 9244 10244 11244 8245 9245 10245 11245 8246 9246 10246 11246 8247 9247 10247 11247 8248 9248 10248 11248 8249 9249 10249 11249 8250 9250 10250 11250 8251 9251 10251 11251 8252 9252 10252 11252 8253 9253 10253 11253 8254 9254 10254 11254 8255 9255 10255 11255 8256 9256 10256 11256 8257 9257 10257 11257 8258 9258 10258 11258 8259 9259 10259 11259 8260 9260 10260 11260 8261 9261 10261 11261 8262 9262 10262 11262 8263 9263 10263 11263 8264 9264 10264 11264 8265 9265 10265 11265 8266 9266 10266 11266 8267 9267 10267 11267 8268 9268 10268 11268 8269 9269 10269 11269 8270 9270 10270 11270 8271 9271 10271 11271 8272 9272 10272 11272 8273 9273 10273 11273 8274 9274 10274 11274 8275 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11320 8321 9321 10321 11321 8322 9322 10322 11322 8323 9323 10323 11323 8324 9324 10324 11324 8325 9325 10325 11325 8326 9326 10326 11326 8327 9327 10327 11327 8328 9328 10328 11328 8329 9329 10329 11329 8330 9330 10330 11330 8331 9331 10331 11331 8332 9332 10332 11332 8333 9333 10333 11333 8334 9334 10334 11334 8335 9335 10335 11335 8336 9336 10336 11336 8337 9337 10337 11337 8338 9338 10338 11338 8339 9339 10339 11339 8340 9340 10340 11340 8341 9341 10341 11341 8342 9342 10342 11342 8343 9343 10343 11343 8344 9344 10344 11344 8345 9345 10345 11345 8346 9346 10346 11346 8347 9347 10347 11347 8348 9348 10348 11348 8349 9349 10349 11349 8350 9350 10350 11350 8351 9351 10351 11351 8352 9352 10352 11352 8353 9353 10353 11353 8354 9354 10354 11354 8355 9355 10355 11355 8356 9356 10356 11356 8357 9357 10357 11357 8358 9358 10358 11358 8359 9359 10359 11359 8360 9360 10360 11360 8361 9361 10361 11361 8362 9362 10362 11362 8363 9363 10363 11363 8364 9364 10364 11364 8365 9365 10365 11365 8366 9366 10366 11366 8367 9367 10367 11367 8368 9368 10368 11368 8369 9369 10369 11369 8370 9370 10370 11370 8371 9371 10371 11371 8372 9372 10372 11372 8373 9373 10373 11373 8374 9374 10374 11374 8375 9375 10375 11375 8376 9376 10376 11376 8377 9377 10377 11377 8378 9378 10378 11378 8379 9379 10379 11379 8380 9380 10380 11380 8381 9381 10381 11381 8382 9382 10382 11382 8383 9383 10383 11383 8384 9384 10384 11384 8385 9385 10385 11385 8386 9386 10386 11386 8387 9387 10387 11387 8388 9388 10388 11388 8389 9389 10389 11389 8390 9390 10390 11390 8391 9391 10391 11391 8392 9392 10392 11392 8393 9393 10393 11393 8394 9394 10394 11394 8395 9395 10395 11395 8396 9396 10396 11396 8397 9397 10397 11397 8398 9398 10398 11398 8399 9399 10399 11399 8400 9400 10400 11400 8401 9401 10401 11401 8402 9402 10402 11402 8403 9403 10403 11403 8404 9404 10404 11404 8405 9405 10405 11405 8406 9406 10406 11406 8407 9407 10407 11407 8408 9408 10408 11408 8409 9409 10409 11409 8410 9410 10410 11410 8411 9411 10411 11411 8412 9412 10412 11412 8413 9413 10413 11413 8414 9414 10414 11414 8415 9415 10415 11415 8416 9416 10416 11416 8417 9417 10417 11417 8418 9418 10418 11418 8419 9419 10419 11419 8420 9420 10420 11420 8421 9421 10421 11421 8422 9422 10422 11422 8423 9423 10423 11423 8424 9424 10424 11424 8425 9425 10425 11425 8426 9426 10426 11426 8427 9427 10427 11427 8428 9428 10428 11428 8429 9429 10429 11429 8430 9430 10430 11430 8431 9431 10431 11431 8432 9432 10432 11432 8433 9433 10433 11433 8434 9434 10434 11434 8435 9435 10435 11435 8436 9436 10436 11436 8437 9437 10437 11437 8438 9438 10438 11438 8439 9439 10439 11439 8440 9440 10440 11440 8441 9441 10441 11441 8442 9442 10442 11442 8443 9443 10443 11443 8444 9444 10444 11444 8445 9445 10445 11445 8446 9446 10446 11446 8447 9447 10447 11447 8448 9448 10448 11448 8449 9449 10449 11449 8450 9450 10450 11450 8451 9451 10451 11451 8452 9452 10452 11452 8453 9453 10453 11453 8454 9454 10454 11454 8455 9455 10455 11455 8456 9456 10456 11456 8457 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In some embodiments, the sequences that specifically bind to cashew comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 12003 to 13002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to cashew may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 13003 to 14002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to cashew may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 14003 to 15002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to cashew may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 15003 to 16002. In one embodiment, the aptamer of the present disclosure that binds to cashew may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 12003 to 16002 listed in Table 5, or variant thereof.

TABLE 5 Aptamer sequences against cashew Aptamer Sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 12003 13003 14003 15003 12004 13004 14004 15004 12005 13005 14005 15005 12006 13006 14006 15006 12007 13007 14007 15007 12008 13008 14008 15008 12009 13009 14009 15009 12010 13010 14010 15010 12011 13011 14011 15011 12012 13012 14012 15012 12013 13013 14013 15013 12014 13014 14014 15014 12015 13015 14015 15015 12016 13016 14016 15016 12017 13017 14017 15017 12018 13018 14018 15018 12019 13019 14019 15019 12020 13020 14020 15020 12021 13021 14021 15021 12022 13022 14022 15022 12023 13023 14023 15023 12024 13024 14024 15024 12025 13025 14025 15025 12026 13026 14026 15026 12027 13027 14027 15027 12028 13028 14028 15028 12029 13029 14029 15029 12030 13030 14030 15030 12031 13031 14031 15031 12032 13032 14032 15032 12033 13033 14033 15033 12034 13034 14034 15034 12035 13035 14035 15035 12036 13036 14036 15036 12037 13037 14037 15037 12038 13038 14038 15038 12039 13039 14039 15039 12040 13040 14040 15040 12041 13041 14041 15041 12042 13042 14042 15042 12043 13043 14043 15043 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14749 15749 12750 13750 14750 15750 12751 13751 14751 15751 12752 13752 14752 15752 12753 13753 14753 15753 12754 13754 14754 15754 12755 13755 14755 15755 12756 13756 14756 15756 12757 13757 14757 15757 12758 13758 14758 15758 12759 13759 14759 15759 12760 13760 14760 15760 12761 13761 14761 15761 12762 13762 14762 15762 12763 13763 14763 15763 12764 13764 14764 15764 12765 13765 14765 15765 12766 13766 14766 15766 12767 13767 14767 15767 12768 13768 14768 15768 12769 13769 14769 15769 12770 13770 14770 15770 12771 13771 14771 15771 12772 13772 14772 15772 12773 13773 14773 15773 12774 13774 14774 15774 12775 13775 14775 15775 12776 13776 14776 15776 12777 13777 14777 15777 12778 13778 14778 15778 12779 13779 14779 15779 12780 13780 14780 15780 12781 13781 14781 15781 12782 13782 14782 15782 12783 13783 14783 15783 12784 13784 14784 15784 12785 13785 14785 15785 12786 13786 14786 15786 12787 13787 14787 15787 12788 13788 14788 15788 12789 13789 14789 15789 12790 13790 14790 15790 12791 13791 14791 15791 12792 13792 14792 15792 12793 13793 14793 15793 12794 13794 14794 15794 12795 13795 14795 15795 12796 13796 14796 15796 12797 13797 14797 15797 12798 13798 14798 15798 12799 13799 14799 15799 12800 13800 14800 15800 12801 13801 14801 15801 12802 13802 14802 15802 12803 13803 14803 15803 12804 13804 14804 15804 12805 13805 14805 15805 12806 13806 14806 15806 12807 13807 14807 15807 12808 13808 14808 15808 12809 13809 14809 15809 12810 13810 14810 15810 12811 13811 14811 15811 12812 13812 14812 15812 12813 13813 14813 15813 12814 13814 14814 15814 12815 13815 14815 15815 12816 13816 14816 15816 12817 13817 14817 15817 12818 13818 14818 15818 12819 13819 14819 15819 12820 13820 14820 15820 12821 13821 14821 15821 12822 13822 14822 15822 12823 13823 14823 15823 12824 13824 14824 15824 12825 13825 14825 15825 12826 13826 14826 15826 12827 13827 14827 15827 12828 13828 14828 15828 12829 13829 14829 15829 12830 13830 14830 15830 12831 13831 14831 15831 12832 13832 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12874 13874 14874 15874 12875 13875 14875 15875 12876 13876 14876 15876 12877 13877 14877 15877 12878 13878 14878 15878 12879 13879 14879 15879 12880 13880 14880 15880 12881 13881 14881 15881 12882 13882 14882 15882 12883 13883 14883 15883 12884 13884 14884 15884 12885 13885 14885 15885 12886 13886 14886 15886 12887 13887 14887 15887 12888 13888 14888 15888 12889 13889 14889 15889 12890 13890 14890 15890 12891 13891 14891 15891 12892 13892 14892 15892 12893 13893 14893 15893 12894 13894 14894 15894 12895 13895 14895 15895 12896 13896 14896 15896 12897 13897 14897 15897 12898 13898 14898 15898 12899 13899 14899 15899 12900 13900 14900 15900 12901 13901 14901 15901 12902 13902 14902 15902 12903 13903 14903 15903 12904 13904 14904 15904 12905 13905 14905 15905 12906 13906 14906 15906 12907 13907 14907 15907 12908 13908 14908 15908 12909 13909 14909 15909 12910 13910 14910 15910 12911 13911 14911 15911 12912 13912 14912 15912 12913 13913 14913 15913 12914 13914 14914 15914 12915 13915 14915 15915 12916 13916 14916 15916 12917 13917 14917 15917 12918 13918 14918 15918 12919 13919 14919 15919 12920 13920 14920 15920 12921 13921 14921 15921 12922 13922 14922 15922 12923 13923 14923 15923 12924 13924 14924 15924 12925 13925 14925 15925 12926 13926 14926 15926 12927 13927 14927 15927 12928 13928 14928 15928 12929 13929 14929 15929 12930 13930 14930 15930 12931 13931 14931 15931 12932 13932 14932 15932 12933 13933 14933 15933 12934 13934 14934 15934 12935 13935 14935 15935 12936 13936 14936 15936 12937 13937 14937 15937 12938 13938 14938 15938 12939 13939 14939 15939 12940 13940 14940 15940 12941 13941 14941 15941 12942 13942 14942 15942 12943 13943 14943 15943 12944 13944 14944 15944 12945 13945 14945 15945 12946 13946 14946 15946 12947 13947 14947 15947 12948 13948 14948 15948 12949 13949 14949 15949 12950 13950 14950 15950 12951 13951 14951 15951 12952 13952 14952 15952 12953 13953 14953 15953 12954 13954 14954 15954 12955 13955 14955 15955 12956 13956 14956 15956 12957 13957 14957 15957 12958 13958 14958 15958 12959 13959 14959 15959 12960 13960 14960 15960 12961 13961 14961 15961 12962 13962 14962 15962 12963 13963 14963 15963 12964 13964 14964 15964 12965 13965 14965 15965 12966 13966 14966 15966 12967 13967 14967 15967 12968 13968 14968 15968 12969 13969 14969 15969 12970 13970 14970 15970 12971 13971 14971 15971 12972 13972 14972 15972 12973 13973 14973 15973 12974 13974 14974 15974 12975 13975 14975 15975 12976 13976 14976 15976 12977 13977 14977 15977 12978 13978 14978 15978 12979 13979 14979 15979 12980 13980 14980 15980 12981 13981 14981 15981 12982 13982 14982 15982 12983 13983 14983 15983 12984 13984 14984 15984 12985 13985 14985 15985 12986 13986 14986 15986 12987 13987 14987 15987 12988 13988 14988 15988 12989 13989 14989 15989 12990 13990 14990 15990 12991 13991 14991 15991 12992 13992 14992 15992 12993 13993 14993 15993 12994 13994 14994 15994 12995 13995 14995 15995 12996 13996 14996 15996 12997 13997 14997 15997 12998 13998 14998 15998 12999 13999 14999 15999 13000 14000 15000 16000 13001 14001 15001 16001 13002 14002 15002 16002

In some embodiments, the sequences that specifically bind to hazelnut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 16003 to 17002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to hazel nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 17003 to 18002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to hazel nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 18003 to 19002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to hazel nut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 19003 to 20002. In one embodiment, the aptamer of the present disclosure that specifically binds to hazelnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 16003 to 20002 listed in Table 6, or variant thereof.

TABLE 6 Aptamer sequences against hazel nut Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 16003 17003 18003 19003 16004 17004 18004 19004 16005 17005 18005 19005 16006 17006 18006 19006 16007 17007 18007 19007 16008 17008 18008 19008 16009 17009 18009 19009 16010 17010 18010 19010 16011 17011 18011 19011 16012 17012 18012 19012 16013 17013 18013 19013 16014 17014 18014 19014 16015 17015 18015 19015 16016 17016 18016 19016 16017 17017 18017 19017 16018 17018 18018 19018 16019 17019 18019 19019 16020 17020 18020 19020 16021 17021 18021 19021 16022 17022 18022 19022 16023 17023 18023 19023 16024 17024 18024 19024 16025 17025 18025 19025 16026 17026 18026 19026 16027 17027 18027 19027 16028 17028 18028 19028 16029 17029 18029 19029 16030 17030 18030 19030 16031 17031 18031 19031 16032 17032 18032 19032 16033 17033 18033 19033 16034 17034 18034 19034 16035 17035 18035 19035 16036 17036 18036 19036 16037 17037 18037 19037 16038 17038 18038 19038 16039 17039 18039 19039 16040 17040 18040 19040 16041 17041 18041 19041 16042 17042 18042 19042 16043 17043 18043 19043 16044 17044 18044 19044 16045 17045 18045 19045 16046 17046 18046 19046 16047 17047 18047 19047 16048 17048 18048 19048 16049 17049 18049 19049 16050 17050 18050 19050 16051 17051 18051 19051 16052 17052 18052 19052 16053 17053 18053 19053 16054 17054 18054 19054 16055 17055 18055 19055 16056 17056 18056 19056 16057 17057 18057 19057 16058 17058 18058 19058 16059 17059 18059 19059 16060 17060 18060 19060 16061 17061 18061 19061 16062 17062 18062 19062 16063 17063 18063 19063 16064 17064 18064 19064 16065 17065 18065 19065 16066 17066 18066 19066 16067 17067 18067 19067 16068 17068 18068 19068 16069 17069 18069 19069 16070 17070 18070 19070 16071 17071 18071 19071 16072 17072 18072 19072 16073 17073 18073 19073 16074 17074 18074 19074 16075 17075 18075 19075 16076 17076 18076 19076 16077 17077 18077 19077 16078 17078 18078 19078 16079 17079 18079 19079 16080 17080 18080 19080 16081 17081 18081 19081 16082 17082 18082 19082 16083 17083 18083 19083 16084 17084 18084 19084 16085 17085 18085 19085 16086 17086 18086 19086 16087 17087 18087 19087 16088 17088 18088 19088 16089 17089 18089 19089 16090 17090 18090 19090 16091 17091 18091 19091 16092 17092 18092 19092 16093 17093 18093 19093 16094 17094 18094 19094 16095 17095 18095 19095 16096 17096 18096 19096 16097 17097 18097 19097 16098 17098 18098 19098 16099 17099 18099 19099 16100 17100 18100 19100 16101 17101 18101 19101 16102 17102 18102 19102 16103 17103 18103 19103 16104 17104 18104 19104 16105 17105 18105 19105 16106 17106 18106 19106 16107 17107 18107 19107 16108 17108 18108 19108 16109 17109 18109 19109 16110 17110 18110 19110 16111 17111 18111 19111 16112 17112 18112 19112 16113 17113 18113 19113 16114 17114 18114 19114 16115 17115 18115 19115 16116 17116 18116 19116 16117 17117 18117 19117 16118 17118 18118 19118 16119 17119 18119 19119 16120 17120 18120 19120 16121 17121 18121 19121 16122 17122 18122 19122 16123 17123 18123 19123 16124 17124 18124 19124 16125 17125 18125 19125 16126 17126 18126 19126 16127 17127 18127 19127 16128 17128 18128 19128 16129 17129 18129 19129 16130 17130 18130 19130 16131 17131 18131 19131 16132 17132 18132 19132 16133 17133 18133 19133 16134 17134 18134 19134 16135 17135 18135 19135 16136 17136 18136 19136 16137 17137 18137 19137 16138 17138 18138 19138 16139 17139 18139 19139 16140 17140 18140 19140 16141 17141 18141 19141 16142 17142 18142 19142 16143 17143 18143 19143 16144 17144 18144 19144 16145 17145 18145 19145 16146 17146 18146 19146 16147 17147 18147 19147 16148 17148 18148 19148 16149 17149 18149 19149 16150 17150 18150 19150 16151 17151 18151 19151 16152 17152 18152 19152 16153 17153 18153 19153 16154 17154 18154 19154 16155 17155 18155 19155 16156 17156 18156 19156 16157 17157 18157 19157 16158 17158 18158 19158 16159 17159 18159 19159 16160 17160 18160 19160 16161 17161 18161 19161 16162 17162 18162 19162 16163 17163 18163 19163 16164 17164 18164 19164 16165 17165 18165 19165 16166 17166 18166 19166 16167 17167 18167 19167 16168 17168 18168 19168 16169 17169 18169 19169 16170 17170 18170 19170 16171 17171 18171 19171 16172 17172 18172 19172 16173 17173 18173 19173 16174 17174 18174 19174 16175 17175 18175 19175 16176 17176 18176 19176 16177 17177 18177 19177 16178 17178 18178 19178 16179 17179 18179 19179 16180 17180 18180 19180 16181 17181 18181 19181 16182 17182 18182 19182 16183 17183 18183 19183 16184 17184 18184 19184 16185 17185 18185 19185 16186 17186 18186 19186 16187 17187 18187 19187 16188 17188 18188 19188 16189 17189 18189 19189 16190 17190 18190 19190 16191 17191 18191 19191 16192 17192 18192 19192 16193 17193 18193 19193 16194 17194 18194 19194 16195 17195 18195 19195 16196 17196 18196 19196 16197 17197 18197 19197 16198 17198 18198 19198 16199 17199 18199 19199 16200 17200 18200 19200 16201 17201 18201 19201 16202 17202 18202 19202 16203 17203 18203 19203 16204 17204 18204 19204 16205 17205 18205 19205 16206 17206 18206 19206 16207 17207 18207 19207 16208 17208 18208 19208 16209 17209 18209 19209 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18749 19749 16750 17750 18750 19750 16751 17751 18751 19751 16752 17752 18752 19752 16753 17753 18753 19753 16754 17754 18754 19754 16755 17755 18755 19755 16756 17756 18756 19756 16757 17757 18757 19757 16758 17758 18758 19758 16759 17759 18759 19759 16760 17760 18760 19760 16761 17761 18761 19761 16762 17762 18762 19762 16763 17763 18763 19763 16764 17764 18764 19764 16765 17765 18765 19765 16766 17766 18766 19766 16767 17767 18767 19767 16768 17768 18768 19768 16769 17769 18769 19769 16770 17770 18770 19770 16771 17771 18771 19771 16772 17772 18772 19772 16773 17773 18773 19773 16774 17774 18774 19774 16775 17775 18775 19775 16776 17776 18776 19776 16777 17777 18777 19777 16778 17778 18778 19778 16779 17779 18779 19779 16780 17780 18780 19780 16781 17781 18781 19781 16782 17782 18782 19782 16783 17783 18783 19783 16784 17784 18784 19784 16785 17785 18785 19785 16786 17786 18786 19786 16787 17787 18787 19787 16788 17788 18788 19788 16789 17789 18789 19789 16790 17790 18790 19790 16791 17791 18791 19791 16792 17792 18792 19792 16793 17793 18793 19793 16794 17794 18794 19794 16795 17795 18795 19795 16796 17796 18796 19796 16797 17797 18797 19797 16798 17798 18798 19798 16799 17799 18799 19799 16800 17800 18800 19800 16801 17801 18801 19801 16802 17802 18802 19802 16803 17803 18803 19803 16804 17804 18804 19804 16805 17805 18805 19805 16806 17806 18806 19806 16807 17807 18807 19807 16808 17808 18808 19808 16809 17809 18809 19809 16810 17810 18810 19810 16811 17811 18811 19811 16812 17812 18812 19812 16813 17813 18813 19813 16814 17814 18814 19814 16815 17815 18815 19815 16816 17816 18816 19816 16817 17817 18817 19817 16818 17818 18818 19818 16819 17819 18819 19819 16820 17820 18820 19820 16821 17821 18821 19821 16822 17822 18822 19822 16823 17823 18823 19823 16824 17824 18824 19824 16825 17825 18825 19825 16826 17826 18826 19826 16827 17827 18827 19827 16828 17828 18828 19828 16829 17829 18829 19829 16830 17830 18830 19830 16831 17831 18831 19831 16832 17832 18832 19832 16833 17833 18833 19833 16834 17834 18834 19834 16835 17835 18835 19835 16836 17836 18836 19836 16837 17837 18837 19837 16838 17838 18838 19838 16839 17839 18839 19839 16840 17840 18840 19840 16841 17841 18841 19841 16842 17842 18842 19842 16843 17843 18843 19843 16844 17844 18844 19844 16845 17845 18845 19845 16846 17846 18846 19846 16847 17847 18847 19847 16848 17848 18848 19848 16849 17849 18849 19849 16850 17850 18850 19850 16851 17851 18851 19851 16852 17852 18852 19852 16853 17853 18853 19853 16854 17854 18854 19854 16855 17855 18855 19855 16856 17856 18856 19856 16857 17857 18857 19857 16858 17858 18858 19858 16859 17859 18859 19859 16860 17860 18860 19860 16861 17861 18861 19861 16862 17862 18862 19862 16863 17863 18863 19863 16864 17864 18864 19864 16865 17865 18865 19865 16866 17866 18866 19866 16867 17867 18867 19867 16868 17868 18868 19868 16869 17869 18869 19869 16870 17870 18870 19870 16871 17871 18871 19871 16872 17872 18872 19872 16873 17873 18873 19873 16874 17874 18874 19874 16875 17875 18875 19875 16876 17876 18876 19876 16877 17877 18877 19877 16878 17878 18878 19878 16879 17879 18879 19879 16880 17880 18880 19880 16881 17881 18881 19881 16882 17882 18882 19882 16883 17883 18883 19883 16884 17884 18884 19884 16885 17885 18885 19885 16886 17886 18886 19886 16887 17887 18887 19887 16888 17888 18888 19888 16889 17889 18889 19889 16890 17890 18890 19890 16891 17891 18891 19891 16892 17892 18892 19892 16893 17893 18893 19893 16894 17894 18894 19894 16895 17895 18895 19895 16896 17896 18896 19896 16897 17897 18897 19897 16898 17898 18898 19898 16899 17899 18899 19899 16900 17900 18900 19900 16901 17901 18901 19901 16902 17902 18902 19902 16903 17903 18903 19903 16904 17904 18904 19904 16905 17905 18905 19905 16906 17906 18906 19906 16907 17907 18907 19907 16908 17908 18908 19908 16909 17909 18909 19909 16910 17910 18910 19910 16911 17911 18911 19911 16912 17912 18912 19912 16913 17913 18913 19913 16914 17914 18914 19914 16915 17915 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16957 17957 18957 19957 16958 17958 18958 19958 16959 17959 18959 19959 16960 17960 18960 19960 16961 17961 18961 19961 16962 17962 18962 19962 16963 17963 18963 19963 16964 17964 18964 19964 16965 17965 18965 19965 16966 17966 18966 19966 16967 17967 18967 19967 16968 17968 18968 19968 16969 17969 18969 19969 16970 17970 18970 19970 16971 17971 18971 19971 16972 17972 18972 19972 16973 17973 18973 19973 16974 17974 18974 19974 16975 17975 18975 19975 16976 17976 18976 19976 16977 17977 18977 19977 16978 17978 18978 19978 16979 17979 18979 19979 16980 17980 18980 19980 16981 17981 18981 19981 16982 17982 18982 19982 16983 17983 18983 19983 16984 17984 18984 19984 16985 17985 18985 19985 16986 17986 18986 19986 16987 17987 18987 19987 16988 17988 18988 19988 16989 17989 18989 19989 16990 17990 18990 19990 16991 17991 18991 19991 16992 17992 18992 19992 16993 17993 18993 19993 16994 17994 18994 19994 16995 17995 18995 19995 16996 17996 18996 19996 16997 17997 18997 19997 16998 17998 18998 19998 16999 17999 18999 19999 17000 18000 19000 20000 17001 18001 19001 20001 17002 18002 19002 20002

In some embodiments, the sequences that specifically bind to pecan comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 20003 to 21002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to pecan may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 21003 to 22002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to pecan may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 22003 to 23002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to pecan may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 23003 to 24002. In one embodiment, the aptamer of the present disclosure that specifically binds to pecan may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 20003 to 24002 listed in Table 7, or variant thereof.

TABLE 7 Aptamer sequences against pecan Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 20003 21003 22003 23003 20004 21004 22004 23004 20005 21005 22005 23005 20006 21006 22006 23006 20007 21007 22007 23007 20008 21008 22008 23008 20009 21009 22009 23009 20010 21010 22010 23010 20011 21011 22011 23011 20012 21012 22012 23012 20013 21013 22013 23013 20014 21014 22014 23014 20015 21015 22015 23015 20016 21016 22016 23016 20017 21017 22017 23017 20018 21018 22018 23018 20019 21019 22019 23019 20020 21020 22020 23020 20021 21021 22021 23021 20022 21022 22022 23022 20023 21023 22023 23023 20024 21024 22024 23024 20025 21025 22025 23025 20026 21026 22026 23026 20027 21027 22027 23027 20028 21028 22028 23028 20029 21029 22029 23029 20030 21030 22030 23030 20031 21031 22031 23031 20032 21032 22032 23032 20033 21033 22033 23033 20034 21034 22034 23034 20035 21035 22035 23035 20036 21036 22036 23036 20037 21037 22037 23037 20038 21038 22038 23038 20039 21039 22039 23039 20040 21040 22040 23040 20041 21041 22041 23041 20042 21042 22042 23042 20043 21043 22043 23043 20044 21044 22044 23044 20045 21045 22045 23045 20046 21046 22046 23046 20047 21047 22047 23047 20048 21048 22048 23048 20049 21049 22049 23049 20050 21050 22050 23050 20051 21051 22051 23051 20052 21052 22052 23052 20053 21053 22053 23053 20054 21054 22054 23054 20055 21055 22055 23055 20056 21056 22056 23056 20057 21057 22057 23057 20058 21058 22058 23058 20059 21059 22059 23059 20060 21060 22060 23060 20061 21061 22061 23061 20062 21062 22062 23062 20063 21063 22063 23063 20064 21064 22064 23064 20065 21065 22065 23065 20066 21066 22066 23066 20067 21067 22067 23067 20068 21068 22068 23068 20069 21069 22069 23069 20070 21070 22070 23070 20071 21071 22071 23071 20072 21072 22072 23072 20073 21073 22073 23073 20074 21074 22074 23074 20075 21075 22075 23075 20076 21076 22076 23076 20077 21077 22077 23077 20078 21078 22078 23078 20079 21079 22079 23079 20080 21080 22080 23080 20081 21081 22081 23081 20082 21082 22082 23082 20083 21083 22083 23083 20084 21084 22084 23084 20085 21085 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20957 21957 22957 23957 20958 21958 22958 23958 20959 21959 22959 23959 20960 21960 22960 23960 20961 21961 22961 23961 20962 21962 22962 23962 20963 21963 22963 23963 20964 21964 22964 23964 20965 21965 22965 23965 20966 21966 22966 23966 20967 21967 22967 23967 20968 21968 22968 23968 20969 21969 22969 23969 20970 21970 22970 23970 20971 21971 22971 23971 20972 21972 22972 23972 20973 21973 22973 23973 20974 21974 22974 23974 20975 21975 22975 23975 20976 21976 22976 23976 20977 21977 22977 23977 20978 21978 22978 23978 20979 21979 22979 23979 20980 21980 22980 23980 20981 21981 22981 23981 20982 21982 22982 23982 20983 21983 22983 23983 20984 21984 22984 23984 20985 21985 22985 23985 20986 21986 22986 23986 20987 21987 22987 23987 20988 21988 22988 23988 20989 21989 22989 23989 20990 21990 22990 23990 20991 21991 22991 23991 20992 21992 22992 23992 20993 21993 22993 23993 20994 21994 22994 23994 20995 21995 22995 23995 20996 21996 22996 23996 20997 21997 22997 23997 20998 21998 22998 23998 20999 21999 22999 23999 21000 22000 23000 24000 21001 22001 23001 24001 21002 22002 23002 24002

In some embodiments, the sequences that specifically bind to pistachio comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 24003 to 25002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to pistachio may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 25003 to 26002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to pistachio may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 26003 to 27002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to pistachio may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 27003 to 28002. In one embodiment, the aptamer of the present disclosure that specifically binds to pistachio may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 24003 to 28002 listed in Table 8, or variant thereof.

TABLE 8 Aptamer sequences against pistachio Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 24003 25003 26003 27003 24004 25004 26004 27004 24005 25005 26005 27005 24006 25006 26006 27006 24007 25007 26007 27007 24008 25008 26008 27008 24009 25009 26009 27009 24010 25010 26010 27010 24011 25011 26011 27011 24012 25012 26012 27012 24013 25013 26013 27013 24014 25014 26014 27014 24015 25015 26015 27015 24016 25016 26016 27016 24017 25017 26017 27017 24018 25018 26018 27018 24019 25019 26019 27019 24020 25020 26020 27020 24021 25021 26021 27021 24022 25022 26022 27022 24023 25023 26023 27023 24024 25024 26024 27024 24025 25025 26025 27025 24026 25026 26026 27026 24027 25027 26027 27027 24028 25028 26028 27028 24029 25029 26029 27029 24030 25030 26030 27030 24031 25031 26031 27031 24032 25032 26032 27032 24033 25033 26033 27033 24034 25034 26034 27034 24035 25035 26035 27035 24036 25036 26036 27036 24037 25037 26037 27037 24038 25038 26038 27038 24039 25039 26039 27039 24040 25040 26040 27040 24041 25041 26041 27041 24042 25042 26042 27042 24043 25043 26043 27043 24044 25044 26044 27044 24045 25045 26045 27045 24046 25046 26046 27046 24047 25047 26047 27047 24048 25048 26048 27048 24049 25049 26049 27049 24050 25050 26050 27050 24051 25051 26051 27051 24052 25052 26052 27052 24053 25053 26053 27053 24054 25054 26054 27054 24055 25055 26055 27055 24056 25056 26056 27056 24057 25057 26057 27057 24058 25058 26058 27058 24059 25059 26059 27059 24060 25060 26060 27060 24061 25061 26061 27061 24062 25062 26062 27062 24063 25063 26063 27063 24064 25064 26064 27064 24065 25065 26065 27065 24066 25066 26066 27066 24067 25067 26067 27067 24068 25068 26068 27068 24069 25069 26069 27069 24070 25070 26070 27070 24071 25071 26071 27071 24072 25072 26072 27072 24073 25073 26073 27073 24074 25074 26074 27074 24075 25075 26075 27075 24076 25076 26076 27076 24077 25077 26077 27077 24078 25078 26078 27078 24079 25079 26079 27079 24080 25080 26080 27080 24081 25081 26081 27081 24082 25082 26082 27082 24083 25083 26083 27083 24084 25084 26084 27084 24085 25085 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26666 27666 24667 25667 26667 27667 24668 25668 26668 27668 24669 25669 26669 27669 24670 25670 26670 27670 24671 25671 26671 27671 24672 25672 26672 27672 24673 25673 26673 27673 24674 25674 26674 27674 24675 25675 26675 27675 24676 25676 26676 27676 24677 25677 26677 27677 24678 25678 26678 27678 24679 25679 26679 27679 24680 25680 26680 27680 24681 25681 26681 27681 24682 25682 26682 27682 24683 25683 26683 27683 24684 25684 26684 27684 24685 25685 26685 27685 24686 25686 26686 27686 24687 25687 26687 27687 24688 25688 26688 27688 24689 25689 26689 27689 24690 25690 26690 27690 24691 25691 26691 27691 24692 25692 26692 27692 24693 25693 26693 27693 24694 25694 26694 27694 24695 25695 26695 27695 24696 25696 26696 27696 24697 25697 26697 27697 24698 25698 26698 27698 24699 25699 26699 27699 24700 25700 26700 27700 24701 25701 26701 27701 24702 25702 26702 27702 24703 25703 26703 27703 24704 25704 26704 27704 24705 25705 26705 27705 24706 25706 26706 27706 24707 25707 26707 27707 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24957 25957 26957 27957 24958 25958 26958 27958 24959 25959 26959 27959 24960 25960 26960 27960 24961 25961 26961 27961 24962 25962 26962 27962 24963 25963 26963 27963 24964 25964 26964 27964 24965 25965 26965 27965 24966 25966 26966 27966 24967 25967 26967 27967 24968 25968 26968 27968 24969 25969 26969 27969 24970 25970 26970 27970 24971 25971 26971 27971 24972 25972 26972 27972 24973 25973 26973 27973 24974 25974 26974 27974 24975 25975 26975 27975 24976 25976 26976 27976 24977 25977 26977 27977 24978 25978 26978 27978 24979 25979 26979 27979 24980 25980 26980 27980 24981 25981 26981 27981 24982 25982 26982 27982 24983 25983 26983 27983 24984 25984 26984 27984 24985 25985 26985 27985 24986 25986 26986 27986 24987 25987 26987 27987 24988 25988 26988 27988 24989 25989 26989 27989 24990 25990 26990 27990 24991 25991 26991 27991 24992 25992 26992 27992 24993 25993 26993 27993 24994 25994 26994 27994 24995 25995 26995 27995 24996 25996 26996 27996 24997 25997 26997 27997 24998 25998 26998 27998 24999 25999 26999 27999 25000 26000 27000 28000 25001 26001 27001 28001 25002 26002 27002 28002

In some embodiments, the sequences that specifically bind to walnut comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 28003 to 29002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to walnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 29003 to 30002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to walnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 30003 to 31002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to walnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 31003 to 32002. In one embodiment, the aptamer of the present disclosure that specifically binds to walnut may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28003 to 32002 listed in Table 9, or variant thereof.

TABLE 9 Aptamer sequences against walnut Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 28003 29003 30003 31003 28004 29004 30004 31004 28005 29005 30005 31005 28006 29006 30006 31006 28007 29007 30007 31007 28008 29008 30008 31008 28009 29009 30009 31009 28010 29010 30010 31010 28011 29011 30011 31011 28012 29012 30012 31012 28013 29013 30013 31013 28014 29014 30014 31014 28015 29015 30015 31015 28016 29016 30016 31016 28017 29017 30017 31017 28018 29018 30018 31018 28019 29019 30019 31019 28020 29020 30020 31020 28021 29021 30021 31021 28022 29022 30022 31022 28023 29023 30023 31023 28024 29024 30024 31024 28025 29025 30025 31025 28026 29026 30026 31026 28027 29027 30027 31027 28028 29028 30028 31028 28029 29029 30029 31029 28030 29030 30030 31030 28031 29031 30031 31031 28032 29032 30032 31032 28033 29033 30033 31033 28034 29034 30034 31034 28035 29035 30035 31035 28036 29036 30036 31036 28037 29037 30037 31037 28038 29038 30038 31038 28039 29039 30039 31039 28040 29040 30040 31040 28041 29041 30041 31041 28042 29042 30042 31042 28043 29043 30043 31043 28044 29044 30044 31044 28045 29045 30045 31045 28046 29046 30046 31046 28047 29047 30047 31047 28048 29048 30048 31048 28049 29049 30049 31049 28050 29050 30050 31050 28051 29051 30051 31051 28052 29052 30052 31052 28053 29053 30053 31053 28054 29054 30054 31054 28055 29055 30055 31055 28056 29056 30056 31056 28057 29057 30057 31057 28058 29058 30058 31058 28059 29059 30059 31059 28060 29060 30060 31060 28061 29061 30061 31061 28062 29062 30062 31062 28063 29063 30063 31063 28064 29064 30064 31064 28065 29065 30065 31065 28066 29066 30066 31066 28067 29067 30067 31067 28068 29068 30068 31068 28069 29069 30069 31069 28070 29070 30070 31070 28071 29071 30071 31071 28072 29072 30072 31072 28073 29073 30073 31073 28074 29074 30074 31074 28075 29075 30075 31075 28076 29076 30076 31076 28077 29077 30077 31077 28078 29078 30078 31078 28079 29079 30079 31079 28080 29080 30080 31080 28081 29081 30081 31081 28082 29082 30082 31082 28083 29083 30083 31083 28084 29084 30084 31084 28085 29085 30085 31085 28086 29086 30086 31086 28087 29087 30087 31087 28088 29088 30088 31088 28089 29089 30089 31089 28090 29090 30090 31090 28091 29091 30091 31091 28092 29092 30092 31092 28093 29093 30093 31093 28094 29094 30094 31094 28095 29095 30095 31095 28096 29096 30096 31096 28097 29097 30097 31097 28098 29098 30098 31098 28099 29099 30099 31099 28100 29100 30100 31100 28101 29101 30101 31101 28102 29102 30102 31102 28103 29103 30103 31103 28104 29104 30104 31104 28105 29105 30105 31105 28106 29106 30106 31106 28107 29107 30107 31107 28108 29108 30108 31108 28109 29109 30109 31109 28110 29110 30110 31110 28111 29111 30111 31111 28112 29112 30112 31112 28113 29113 30113 31113 28114 29114 30114 31114 28115 29115 30115 31115 28116 29116 30116 31116 28117 29117 30117 31117 28118 29118 30118 31118 28119 29119 30119 31119 28120 29120 30120 31120 28121 29121 30121 31121 28122 29122 30122 31122 28123 29123 30123 31123 28124 29124 30124 31124 28125 29125 30125 31125 28126 29126 30126 31126 28127 29127 30127 31127 28128 29128 30128 31128 28129 29129 30129 31129 28130 29130 30130 31130 28131 29131 30131 31131 28132 29132 30132 31132 28133 29133 30133 31133 28134 29134 30134 31134 28135 29135 30135 31135 28136 29136 30136 31136 28137 29137 30137 31137 28138 29138 30138 31138 28139 29139 30139 31139 28140 29140 30140 31140 28141 29141 30141 31141 28142 29142 30142 31142 28143 29143 30143 31143 28144 29144 30144 31144 28145 29145 30145 31145 28146 29146 30146 31146 28147 29147 30147 31147 28148 29148 30148 31148 28149 29149 30149 31149 28150 29150 30150 31150 28151 29151 30151 31151 28152 29152 30152 31152 28153 29153 30153 31153 28154 29154 30154 31154 28155 29155 30155 31155 28156 29156 30156 31156 28157 29157 30157 31157 28158 29158 30158 31158 28159 29159 30159 31159 28160 29160 30160 31160 28161 29161 30161 31161 28162 29162 30162 31162 28163 29163 30163 31163 28164 29164 30164 31164 28165 29165 30165 31165 28166 29166 30166 31166 28167 29167 30167 31167 28168 29168 30168 31168 28169 29169 30169 31169 28170 29170 30170 31170 28171 29171 30171 31171 28172 29172 30172 31172 28173 29173 30173 31173 28174 29174 30174 31174 28175 29175 30175 31175 28176 29176 30176 31176 28177 29177 30177 31177 28178 29178 30178 31178 28179 29179 30179 31179 28180 29180 30180 31180 28181 29181 30181 31181 28182 29182 30182 31182 28183 29183 30183 31183 28184 29184 30184 31184 28185 29185 30185 31185 28186 29186 30186 31186 28187 29187 30187 31187 28188 29188 30188 31188 28189 29189 30189 31189 28190 29190 30190 31190 28191 29191 30191 31191 28192 29192 30192 31192 28193 29193 30193 31193 28194 29194 30194 31194 28195 29195 30195 31195 28196 29196 30196 31196 28197 29197 30197 31197 28198 29198 30198 31198 28199 29199 30199 31199 28200 29200 30200 31200 28201 29201 30201 31201 28202 29202 30202 31202 28203 29203 30203 31203 28204 29204 30204 31204 28205 29205 30205 31205 28206 29206 30206 31206 28207 29207 30207 31207 28208 29208 30208 31208 28209 29209 30209 31209 28210 29210 30210 31210 28211 29211 30211 31211 28212 29212 30212 31212 28213 29213 30213 31213 28214 29214 30214 31214 28215 29215 30215 31215 28216 29216 30216 31216 28217 29217 30217 31217 28218 29218 30218 31218 28219 29219 30219 31219 28220 29220 30220 31220 28221 29221 30221 31221 28222 29222 30222 31222 28223 29223 30223 31223 28224 29224 30224 31224 28225 29225 30225 31225 28226 29226 30226 31226 28227 29227 30227 31227 28228 29228 30228 31228 28229 29229 30229 31229 28230 29230 30230 31230 28231 29231 30231 31231 28232 29232 30232 31232 28233 29233 30233 31233 28234 29234 30234 31234 28235 29235 30235 31235 28236 29236 30236 31236 28237 29237 30237 31237 28238 29238 30238 31238 28239 29239 30239 31239 28240 29240 30240 31240 28241 29241 30241 31241 28242 29242 30242 31242 28243 29243 30243 31243 28244 29244 30244 31244 28245 29245 30245 31245 28246 29246 30246 31246 28247 29247 30247 31247 28248 29248 30248 31248 28249 29249 30249 31249 28250 29250 30250 31250 28251 29251 30251 31251 28252 29252 30252 31252 28253 29253 30253 31253 28254 29254 30254 31254 28255 29255 30255 31255 28256 29256 30256 31256 28257 29257 30257 31257 28258 29258 30258 31258 28259 29259 30259 31259 28260 29260 30260 31260 28261 29261 30261 31261 28262 29262 30262 31262 28263 29263 30263 31263 28264 29264 30264 31264 28265 29265 30265 31265 28266 29266 30266 31266 28267 29267 30267 31267 28268 29268 30268 31268 28269 29269 30269 31269 28270 29270 30270 31270 28271 29271 30271 31271 28272 29272 30272 31272 28273 29273 30273 31273 28274 29274 30274 31274 28275 29275 30275 31275 28276 29276 30276 31276 28277 29277 30277 31277 28278 29278 30278 31278 28279 29279 30279 31279 28280 29280 30280 31280 28281 29281 30281 31281 28282 29282 30282 31282 28283 29283 30283 31283 28284 29284 30284 31284 28285 29285 30285 31285 28286 29286 30286 31286 28287 29287 30287 31287 28288 29288 30288 31288 28289 29289 30289 31289 28290 29290 30290 31290 28291 29291 30291 31291 28292 29292 30292 31292 28293 29293 30293 31293 28294 29294 30294 31294 28295 29295 30295 31295 28296 29296 30296 31296 28297 29297 30297 31297 28298 29298 30298 31298 28299 29299 30299 31299 28300 29300 30300 31300 28301 29301 30301 31301 28302 29302 30302 31302 28303 29303 30303 31303 28304 29304 30304 31304 28305 29305 30305 31305 28306 29306 30306 31306 28307 29307 30307 31307 28308 29308 30308 31308 28309 29309 30309 31309 28310 29310 30310 31310 28311 29311 30311 31311 28312 29312 30312 31312 28313 29313 30313 31313 28314 29314 30314 31314 28315 29315 30315 31315 28316 29316 30316 31316 28317 29317 30317 31317 28318 29318 30318 31318 28319 29319 30319 31319 28320 29320 30320 31320 28321 29321 30321 31321 28322 29322 30322 31322 28323 29323 30323 31323 28324 29324 30324 31324 28325 29325 30325 31325 28326 29326 30326 31326 28327 29327 30327 31327 28328 29328 30328 31328 28329 29329 30329 31329 28330 29330 30330 31330 28331 29331 30331 31331 28332 29332 30332 31332 28333 29333 30333 31333 28334 29334 30334 31334 28335 29335 30335 31335 28336 29336 30336 31336 28337 29337 30337 31337 28338 29338 30338 31338 28339 29339 30339 31339 28340 29340 30340 31340 28341 29341 30341 31341 28342 29342 30342 31342 28343 29343 30343 31343 28344 29344 30344 31344 28345 29345 30345 31345 28346 29346 30346 31346 28347 29347 30347 31347 28348 29348 30348 31348 28349 29349 30349 31349 28350 29350 30350 31350 28351 29351 30351 31351 28352 29352 30352 31352 28353 29353 30353 31353 28354 29354 30354 31354 28355 29355 30355 31355 28356 29356 30356 31356 28357 29357 30357 31357 28358 29358 30358 31358 28359 29359 30359 31359 28360 29360 30360 31360 28361 29361 30361 31361 28362 29362 30362 31362 28363 29363 30363 31363 28364 29364 30364 31364 28365 29365 30365 31365 28366 29366 30366 31366 28367 29367 30367 31367 28368 29368 30368 31368 28369 29369 30369 31369 28370 29370 30370 31370 28371 29371 30371 31371 28372 29372 30372 31372 28373 29373 30373 31373 28374 29374 30374 31374 28375 29375 30375 31375 28376 29376 30376 31376 28377 29377 30377 31377 28378 29378 30378 31378 28379 29379 30379 31379 28380 29380 30380 31380 28381 29381 30381 31381 28382 29382 30382 31382 28383 29383 30383 31383 28384 29384 30384 31384 28385 29385 30385 31385 28386 29386 30386 31386 28387 29387 30387 31387 28388 29388 30388 31388 28389 29389 30389 31389 28390 29390 30390 31390 28391 29391 30391 31391 28392 29392 30392 31392 28393 29393 30393 31393 28394 29394 30394 31394 28395 29395 30395 31395 28396 29396 30396 31396 28397 29397 30397 31397 28398 29398 30398 31398 28399 29399 30399 31399 28400 29400 30400 31400 28401 29401 30401 31401 28402 29402 30402 31402 28403 29403 30403 31403 28404 29404 30404 31404 28405 29405 30405 31405 28406 29406 30406 31406 28407 29407 30407 31407 28408 29408 30408 31408 28409 29409 30409 31409 28410 29410 30410 31410 28411 29411 30411 31411 28412 29412 30412 31412 28413 29413 30413 31413 28414 29414 30414 31414 28415 29415 30415 31415 28416 29416 30416 31416 28417 29417 30417 31417 28418 29418 30418 31418 28419 29419 30419 31419 28420 29420 30420 31420 28421 29421 30421 31421 28422 29422 30422 31422 28423 29423 30423 31423 28424 29424 30424 31424 28425 29425 30425 31425 28426 29426 30426 31426 28427 29427 30427 31427 28428 29428 30428 31428 28429 29429 30429 31429 28430 29430 30430 31430 28431 29431 30431 31431 28432 29432 30432 31432 28433 29433 30433 31433 28434 29434 30434 31434 28435 29435 30435 31435 28436 29436 30436 31436 28437 29437 30437 31437 28438 29438 30438 31438 28439 29439 30439 31439 28440 29440 30440 31440 28441 29441 30441 31441 28442 29442 30442 31442 28443 29443 30443 31443 28444 29444 30444 31444 28445 29445 30445 31445 28446 29446 30446 31446 28447 29447 30447 31447 28448 29448 30448 31448 28449 29449 30449 31449 28450 29450 30450 31450 28451 29451 30451 31451 28452 29452 30452 31452 28453 29453 30453 31453 28454 29454 30454 31454 28455 29455 30455 31455 28456 29456 30456 31456 28457 29457 30457 31457 28458 29458 30458 31458 28459 29459 30459 31459 28460 29460 30460 31460 28461 29461 30461 31461 28462 29462 30462 31462 28463 29463 30463 31463 28464 29464 30464 31464 28465 29465 30465 31465 28466 29466 30466 31466 28467 29467 30467 31467 28468 29468 30468 31468 28469 29469 30469 31469 28470 29470 30470 31470 28471 29471 30471 31471 28472 29472 30472 31472 28473 29473 30473 31473 28474 29474 30474 31474 28475 29475 30475 31475 28476 29476 30476 31476 28477 29477 30477 31477 28478 29478 30478 31478 28479 29479 30479 31479 28480 29480 30480 31480 28481 29481 30481 31481 28482 29482 30482 31482 28483 29483 30483 31483 28484 29484 30484 31484 28485 29485 30485 31485 28486 29486 30486 31486 28487 29487 30487 31487 28488 29488 30488 31488 28489 29489 30489 31489 28490 29490 30490 31490 28491 29491 30491 31491 28492 29492 30492 31492 28493 29493 30493 31493 28494 29494 30494 31494 28495 29495 30495 31495 28496 29496 30496 31496 28497 29497 30497 31497 28498 29498 30498 31498 28499 29499 30499 31499 28500 29500 30500 31500 28501 29501 30501 31501 28502 29502 30502 31502 28503 29503 30503 31503 28504 29504 30504 31504 28505 29505 30505 31505 28506 29506 30506 31506 28507 29507 30507 31507 28508 29508 30508 31508 28509 29509 30509 31509 28510 29510 30510 31510 28511 29511 30511 31511 28512 29512 30512 31512 28513 29513 30513 31513 28514 29514 30514 31514 28515 29515 30515 31515 28516 29516 30516 31516 28517 29517 30517 31517 28518 29518 30518 31518 28519 29519 30519 31519 28520 29520 30520 31520 28521 29521 30521 31521 28522 29522 30522 31522 28523 29523 30523 31523 28524 29524 30524 31524 28525 29525 30525 31525 28526 29526 30526 31526 28527 29527 30527 31527 28528 29528 30528 31528 28529 29529 30529 31529 28530 29530 30530 31530 28531 29531 30531 31531 28532 29532 30532 31532 28533 29533 30533 31533 28534 29534 30534 31534 28535 29535 30535 31535 28536 29536 30536 31536 28537 29537 30537 31537 28538 29538 30538 31538 28539 29539 30539 31539 28540 29540 30540 31540 28541 29541 30541 31541 28542 29542 30542 31542 28543 29543 30543 31543 28544 29544 30544 31544 28545 29545 30545 31545 28546 29546 30546 31546 28547 29547 30547 31547 28548 29548 30548 31548 28549 29549 30549 31549 28550 29550 30550 31550 28551 29551 30551 31551 28552 29552 30552 31552 28553 29553 30553 31553 28554 29554 30554 31554 28555 29555 30555 31555 28556 29556 30556 31556 28557 29557 30557 31557 28558 29558 30558 31558 28559 29559 30559 31559 28560 29560 30560 31560 28561 29561 30561 31561 28562 29562 30562 31562 28563 29563 30563 31563 28564 29564 30564 31564 28565 29565 30565 31565 28566 29566 30566 31566 28567 29567 30567 31567 28568 29568 30568 31568 28569 29569 30569 31569 28570 29570 30570 31570 28571 29571 30571 31571 28572 29572 30572 31572 28573 29573 30573 31573 28574 29574 30574 31574 28575 29575 30575 31575 28576 29576 30576 31576 28577 29577 30577 31577 28578 29578 30578 31578 28579 29579 30579 31579 28580 29580 30580 31580 28581 29581 30581 31581 28582 29582 30582 31582 28583 29583 30583 31583 28584 29584 30584 31584 28585 29585 30585 31585 28586 29586 30586 31586 28587 29587 30587 31587 28588 29588 30588 31588 28589 29589 30589 31589 28590 29590 30590 31590 28591 29591 30591 31591 28592 29592 30592 31592 28593 29593 30593 31593 28594 29594 30594 31594 28595 29595 30595 31595 28596 29596 30596 31596 28597 29597 30597 31597 28598 29598 30598 31598 28599 29599 30599 31599 28600 29600 30600 31600 28601 29601 30601 31601 28602 29602 30602 31602 28603 29603 30603 31603 28604 29604 30604 31604 28605 29605 30605 31605 28606 29606 30606 31606 28607 29607 30607 31607 28608 29608 30608 31608 28609 29609 30609 31609 28610 29610 30610 31610 28611 29611 30611 31611 28612 29612 30612 31612 28613 29613 30613 31613 28614 29614 30614 31614 28615 29615 30615 31615 28616 29616 30616 31616 28617 29617 30617 31617 28618 29618 30618 31618 28619 29619 30619 31619 28620 29620 30620 31620 28621 29621 30621 31621 28622 29622 30622 31622 28623 29623 30623 31623 28624 29624 30624 31624 28625 29625 30625 31625 28626 29626 30626 31626 28627 29627 30627 31627 28628 29628 30628 31628 28629 29629 30629 31629 28630 29630 30630 31630 28631 29631 30631 31631 28632 29632 30632 31632 28633 29633 30633 31633 28634 29634 30634 31634 28635 29635 30635 31635 28636 29636 30636 31636 28637 29637 30637 31637 28638 29638 30638 31638 28639 29639 30639 31639 28640 29640 30640 31640 28641 29641 30641 31641 28642 29642 30642 31642 28643 29643 30643 31643 28644 29644 30644 31644 28645 29645 30645 31645 28646 29646 30646 31646 28647 29647 30647 31647 28648 29648 30648 31648 28649 29649 30649 31649 28650 29650 30650 31650 28651 29651 30651 31651 28652 29652 30652 31652 28653 29653 30653 31653 28654 29654 30654 31654 28655 29655 30655 31655 28656 29656 30656 31656 28657 29657 30657 31657 28658 29658 30658 31658 28659 29659 30659 31659 28660 29660 30660 31660 28661 29661 30661 31661 28662 29662 30662 31662 28663 29663 30663 31663 28664 29664 30664 31664 28665 29665 30665 31665 28666 29666 30666 31666 28667 29667 30667 31667 28668 29668 30668 31668 28669 29669 30669 31669 28670 29670 30670 31670 28671 29671 30671 31671 28672 29672 30672 31672 28673 29673 30673 31673 28674 29674 30674 31674 28675 29675 30675 31675 28676 29676 30676 31676 28677 29677 30677 31677 28678 29678 30678 31678 28679 29679 30679 31679 28680 29680 30680 31680 28681 29681 30681 31681 28682 29682 30682 31682 28683 29683 30683 31683 28684 29684 30684 31684 28685 29685 30685 31685 28686 29686 30686 31686 28687 29687 30687 31687 28688 29688 30688 31688 28689 29689 30689 31689 28690 29690 30690 31690 28691 29691 30691 31691 28692 29692 30692 31692 28693 29693 30693 31693 28694 29694 30694 31694 28695 29695 30695 31695 28696 29696 30696 31696 28697 29697 30697 31697 28698 29698 30698 31698 28699 29699 30699 31699 28700 29700 30700 31700 28701 29701 30701 31701 28702 29702 30702 31702 28703 29703 30703 31703 28704 29704 30704 31704 28705 29705 30705 31705 28706 29706 30706 31706 28707 29707 30707 31707 28708 29708 30708 31708 28709 29709 30709 31709 28710 29710 30710 31710 28711 29711 30711 31711 28712 29712 30712 31712 28713 29713 30713 31713 28714 29714 30714 31714 28715 29715 30715 31715 28716 29716 30716 31716 28717 29717 30717 31717 28718 29718 30718 31718 28719 29719 30719 31719 28720 29720 30720 31720 28721 29721 30721 31721 28722 29722 30722 31722 28723 29723 30723 31723 28724 29724 30724 31724 28725 29725 30725 31725 28726 29726 30726 31726 28727 29727 30727 31727 28728 29728 30728 31728 28729 29729 30729 31729 28730 29730 30730 31730 28731 29731 30731 31731 28732 29732 30732 31732 28733 29733 30733 31733 28734 29734 30734 31734 28735 29735 30735 31735 28736 29736 30736 31736 28737 29737 30737 31737 28738 29738 30738 31738 28739 29739 30739 31739 28740 29740 30740 31740 28741 29741 30741 31741 28742 29742 30742 31742 28743 29743 30743 31743 28744 29744 30744 31744 28745 29745 30745 31745 28746 29746 30746 31746 28747 29747 30747 31747 28748 29748 30748 31748 28749 29749 30749 31749 28750 29750 30750 31750 28751 29751 30751 31751 28752 29752 30752 31752 28753 29753 30753 31753 28754 29754 30754 31754 28755 29755 30755 31755 28756 29756 30756 31756 28757 29757 30757 31757 28758 29758 30758 31758 28759 29759 30759 31759 28760 29760 30760 31760 28761 29761 30761 31761 28762 29762 30762 31762 28763 29763 30763 31763 28764 29764 30764 31764 28765 29765 30765 31765 28766 29766 30766 31766 28767 29767 30767 31767 28768 29768 30768 31768 28769 29769 30769 31769 28770 29770 30770 31770 28771 29771 30771 31771 28772 29772 30772 31772 28773 29773 30773 31773 28774 29774 30774 31774 28775 29775 30775 31775 28776 29776 30776 31776 28777 29777 30777 31777 28778 29778 30778 31778 28779 29779 30779 31779 28780 29780 30780 31780 28781 29781 30781 31781 28782 29782 30782 31782 28783 29783 30783 31783 28784 29784 30784 31784 28785 29785 30785 31785 28786 29786 30786 31786 28787 29787 30787 31787 28788 29788 30788 31788 28789 29789 30789 31789 28790 29790 30790 31790 28791 29791 30791 31791 28792 29792 30792 31792 28793 29793 30793 31793 28794 29794 30794 31794 28795 29795 30795 31795 28796 29796 30796 31796 28797 29797 30797 31797 28798 29798 30798 31798 28799 29799 30799 31799 28800 29800 30800 31800 28801 29801 30801 31801 28802 29802 30802 31802 28803 29803 30803 31803 28804 29804 30804 31804 28805 29805 30805 31805 28806 29806 30806 31806 28807 29807 30807 31807 28808 29808 30808 31808 28809 29809 30809 31809 28810 29810 30810 31810 28811 29811 30811 31811 28812 29812 30812 31812 28813 29813 30813 31813 28814 29814 30814 31814 28815 29815 30815 31815 28816 29816 30816 31816 28817 29817 30817 31817 28818 29818 30818 31818 28819 29819 30819 31819 28820 29820 30820 31820 28821 29821 30821 31821 28822 29822 30822 31822 28823 29823 30823 31823 28824 29824 30824 31824 28825 29825 30825 31825 28826 29826 30826 31826 28827 29827 30827 31827 28828 29828 30828 31828 28829 29829 30829 31829 28830 29830 30830 31830 28831 29831 30831 31831 28832 29832 30832 31832 28833 29833 30833 31833 28834 29834 30834 31834 28835 29835 30835 31835 28836 29836 30836 31836 28837 29837 30837 31837 28838 29838 30838 31838 28839 29839 30839 31839 28840 29840 30840 31840 28841 29841 30841 31841 28842 29842 30842 31842 28843 29843 30843 31843 28844 29844 30844 31844 28845 29845 30845 31845 28846 29846 30846 31846 28847 29847 30847 31847 28848 29848 30848 31848 28849 29849 30849 31849 28850 29850 30850 31850 28851 29851 30851 31851 28852 29852 30852 31852 28853 29853 30853 31853 28854 29854 30854 31854 28855 29855 30855 31855 28856 29856 30856 31856 28857 29857 30857 31857 28858 29858 30858 31858 28859 29859 30859 31859 28860 29860 30860 31860 28861 29861 30861 31861 28862 29862 30862 31862 28863 29863 30863 31863 28864 29864 30864 31864 28865 29865 30865 31865 28866 29866 30866 31866 28867 29867 30867 31867 28868 29868 30868 31868 28869 29869 30869 31869 28870 29870 30870 31870 28871 29871 30871 31871 28872 29872 30872 31872 28873 29873 30873 31873 28874 29874 30874 31874 28875 29875 30875 31875 28876 29876 30876 31876 28877 29877 30877 31877 28878 29878 30878 31878 28879 29879 30879 31879 28880 29880 30880 31880 28881 29881 30881 31881 28882 29882 30882 31882 28883 29883 30883 31883 28884 29884 30884 31884 28885 29885 30885 31885 28886 29886 30886 31886 28887 29887 30887 31887 28888 29888 30888 31888 28889 29889 30889 31889 28890 29890 30890 31890 28891 29891 30891 31891 28892 29892 30892 31892 28893 29893 30893 31893 28894 29894 30894 31894 28895 29895 30895 31895 28896 29896 30896 31896 28897 29897 30897 31897 28898 29898 30898 31898 28899 29899 30899 31899 28900 29900 30900 31900 28901 29901 30901 31901 28902 29902 30902 31902 28903 29903 30903 31903 28904 29904 30904 31904 28905 29905 30905 31905 28906 29906 30906 31906 28907 29907 30907 31907 28908 29908 30908 31908 28909 29909 30909 31909 28910 29910 30910 31910 28911 29911 30911 31911 28912 29912 30912 31912 28913 29913 30913 31913 28914 29914 30914 31914 28915 29915 30915 31915 28916 29916 30916 31916 28917 29917 30917 31917 28918 29918 30918 31918 28919 29919 30919 31919 28920 29920 30920 31920 28921 29921 30921 31921 28922 29922 30922 31922 28923 29923 30923 31923 28924 29924 30924 31924 28925 29925 30925 31925 28926 29926 30926 31926 28927 29927 30927 31927 28928 29928 30928 31928 28929 29929 30929 31929 28930 29930 30930 31930 28931 29931 30931 31931 28932 29932 30932 31932 28933 29933 30933 31933 28934 29934 30934 31934 28935 29935 30935 31935 28936 29936 30936 31936 28937 29937 30937 31937 28938 29938 30938 31938 28939 29939 30939 31939 28940 29940 30940 31940 28941 29941 30941 31941 28942 29942 30942 31942 28943 29943 30943 31943 28944 29944 30944 31944 28945 29945 30945 31945 28946 29946 30946 31946 28947 29947 30947 31947 28948 29948 30948 31948 28949 29949 30949 31949 28950 29950 30950 31950 28951 29951 30951 31951 28952 29952 30952 31952 28953 29953 30953 31953 28954 29954 30954 31954 28955 29955 30955 31955 28956 29956 30956 31956 28957 29957 30957 31957 28958 29958 30958 31958 28959 29959 30959 31959 28960 29960 30960 31960 28961 29961 30961 31961 28962 29962 30962 31962 28963 29963 30963 31963 28964 29964 30964 31964 28965 29965 30965 31965 28966 29966 30966 31966 28967 29967 30967 31967 28968 29968 30968 31968 28969 29969 30969 31969 28970 29970 30970 31970 28971 29971 30971 31971 28972 29972 30972 31972 28973 29973 30973 31973 28974 29974 30974 31974 28975 29975 30975 31975 28976 29976 30976 31976 28977 29977 30977 31977 28978 29978 30978 31978 28979 29979 30979 31979 28980 29980 30980 31980 28981 29981 30981 31981 28982 29982 30982 31982 28983 29983 30983 31983 28984 29984 30984 31984 28985 29985 30985 31985 28986 29986 30986 31986 28987 29987 30987 31987 28988 29988 30988 31988 28989 29989 30989 31989 28990 29990 30990 31990 28991 29991 30991 31991 28992 29992 30992 31992 28993 29993 30993 31993 28994 29994 30994 31994 28995 29995 30995 31995 28996 29996 30996 31996 28997 29997 30997 31997 28998 29998 30998 31998 28999 29999 30999 31999 29000 30000 31000 32000 29001 30001 31001 32001 29002 30002 31002 32002

In some embodiments, the sequences that specifically bind to all nuts comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 32003 to 33002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to all nuts may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 33003 to 34002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to all nuts may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 34003 to 35002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to all nuts may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 35003 to 36002. In one embodiment, the aptamer of the present disclosure that can bind to all nuts may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 32003 to 36002 listed in Table 10, or variant thereof.

TABLE 10 Aptamer sequences against all nuts Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 32003 33003 34003 35003 32004 33004 34004 35004 32005 33005 34005 35005 32006 33006 34006 35006 32007 33007 34007 35007 32008 33008 34008 35008 32009 33009 34009 35009 32010 33010 34010 35010 32011 33011 34011 35011 32012 33012 34012 35012 32013 33013 34013 35013 32014 33014 34014 35014 32015 33015 34015 35015 32016 33016 34016 35016 32017 33017 34017 35017 32018 33018 34018 35018 32019 33019 34019 35019 32020 33020 34020 35020 32021 33021 34021 35021 32022 33022 34022 35022 32023 33023 34023 35023 32024 33024 34024 35024 32025 33025 34025 35025 32026 33026 34026 35026 32027 33027 34027 35027 32028 33028 34028 35028 32029 33029 34029 35029 32030 33030 34030 35030 32031 33031 34031 35031 32032 33032 34032 35032 32033 33033 34033 35033 32034 33034 34034 35034 32035 33035 34035 35035 32036 33036 34036 35036 32037 33037 34037 35037 32038 33038 34038 35038 32039 33039 34039 35039 32040 33040 34040 35040 32041 33041 34041 35041 32042 33042 34042 35042 32043 33043 34043 35043 32044 33044 34044 35044 32045 33045 34045 35045 32046 33046 34046 35046 32047 33047 34047 35047 32048 33048 34048 35048 32049 33049 34049 35049 32050 33050 34050 35050 32051 33051 34051 35051 32052 33052 34052 35052 32053 33053 34053 35053 32054 33054 34054 35054 32055 33055 34055 35055 32056 33056 34056 35056 32057 33057 34057 35057 32058 33058 34058 35058 32059 33059 34059 35059 32060 33060 34060 35060 32061 33061 34061 35061 32062 33062 34062 35062 32063 33063 34063 35063 32064 33064 34064 35064 32065 33065 34065 35065 32066 33066 34066 35066 32067 33067 34067 35067 32068 33068 34068 35068 32069 33069 34069 35069 32070 33070 34070 35070 32071 33071 34071 35071 32072 33072 34072 35072 32073 33073 34073 35073 32074 33074 34074 35074 32075 33075 34075 35075 32076 33076 34076 35076 32077 33077 34077 35077 32078 33078 34078 35078 32079 33079 34079 35079 32080 33080 34080 35080 32081 33081 34081 35081 32082 33082 34082 35082 32083 33083 34083 35083 32084 33084 34084 35084 32085 33085 34085 35085 32086 33086 34086 35086 32087 33087 34087 35087 32088 33088 34088 35088 32089 33089 34089 35089 32090 33090 34090 35090 32091 33091 34091 35091 32092 33092 34092 35092 32093 33093 34093 35093 32094 33094 34094 35094 32095 33095 34095 35095 32096 33096 34096 35096 32097 33097 34097 35097 32098 33098 34098 35098 32099 33099 34099 35099 32100 33100 34100 35100 32101 33101 34101 35101 32102 33102 34102 35102 32103 33103 34103 35103 32104 33104 34104 35104 32105 33105 34105 35105 32106 33106 34106 35106 32107 33107 34107 35107 32108 33108 34108 35108 32109 33109 34109 35109 32110 33110 34110 35110 32111 33111 34111 35111 32112 33112 34112 35112 32113 33113 34113 35113 32114 33114 34114 35114 32115 33115 34115 35115 32116 33116 34116 35116 32117 33117 34117 35117 32118 33118 34118 35118 32119 33119 34119 35119 32120 33120 34120 35120 32121 33121 34121 35121 32122 33122 34122 35122 32123 33123 34123 35123 32124 33124 34124 35124 32125 33125 34125 35125 32126 33126 34126 35126 32127 33127 34127 35127 32128 33128 34128 35128 32129 33129 34129 35129 32130 33130 34130 35130 32131 33131 34131 35131 32132 33132 34132 35132 32133 33133 34133 35133 32134 33134 34134 35134 32135 33135 34135 35135 32136 33136 34136 35136 32137 33137 34137 35137 32138 33138 34138 35138 32139 33139 34139 35139 32140 33140 34140 35140 32141 33141 34141 35141 32142 33142 34142 35142 32143 33143 34143 35143 32144 33144 34144 35144 32145 33145 34145 35145 32146 33146 34146 35146 32147 33147 34147 35147 32148 33148 34148 35148 32149 33149 34149 35149 32150 33150 34150 35150 32151 33151 34151 35151 32152 33152 34152 35152 32153 33153 34153 35153 32154 33154 34154 35154 32155 33155 34155 35155 32156 33156 34156 35156 32157 33157 34157 35157 32158 33158 34158 35158 32159 33159 34159 35159 32160 33160 34160 35160 32161 33161 34161 35161 32162 33162 34162 35162 32163 33163 34163 35163 32164 33164 34164 35164 32165 33165 34165 35165 32166 33166 34166 35166 32167 33167 34167 35167 32168 33168 34168 35168 32169 33169 34169 35169 32170 33170 34170 35170 32171 33171 34171 35171 32172 33172 34172 35172 32173 33173 34173 35173 32174 33174 34174 35174 32175 33175 34175 35175 32176 33176 34176 35176 32177 33177 34177 35177 32178 33178 34178 35178 32179 33179 34179 35179 32180 33180 34180 35180 32181 33181 34181 35181 32182 33182 34182 35182 32183 33183 34183 35183 32184 33184 34184 35184 32185 33185 34185 35185 32186 33186 34186 35186 32187 33187 34187 35187 32188 33188 34188 35188 32189 33189 34189 35189 32190 33190 34190 35190 32191 33191 34191 35191 32192 33192 34192 35192 32193 33193 34193 35193 32194 33194 34194 35194 32195 33195 34195 35195 32196 33196 34196 35196 32197 33197 34197 35197 32198 33198 34198 35198 32199 33199 34199 35199 32200 33200 34200 35200 32201 33201 34201 35201 32202 33202 34202 35202 32203 33203 34203 35203 32204 33204 34204 35204 32205 33205 34205 35205 32206 33206 34206 35206 32207 33207 34207 35207 32208 33208 34208 35208 32209 33209 34209 35209 32210 33210 34210 35210 32211 33211 34211 35211 32212 33212 34212 35212 32213 33213 34213 35213 32214 33214 34214 35214 32215 33215 34215 35215 32216 33216 34216 35216 32217 33217 34217 35217 32218 33218 34218 35218 32219 33219 34219 35219 32220 33220 34220 35220 32221 33221 34221 35221 32222 33222 34222 35222 32223 33223 34223 35223 32224 33224 34224 35224 32225 33225 34225 35225 32226 33226 34226 35226 32227 33227 34227 35227 32228 33228 34228 35228 32229 33229 34229 35229 32230 33230 34230 35230 32231 33231 34231 35231 32232 33232 34232 35232 32233 33233 34233 35233 32234 33234 34234 35234 32235 33235 34235 35235 32236 33236 34236 35236 32237 33237 34237 35237 32238 33238 34238 35238 32239 33239 34239 35239 32240 33240 34240 35240 32241 33241 34241 35241 32242 33242 34242 35242 32243 33243 34243 35243 32244 33244 34244 35244 32245 33245 34245 35245 32246 33246 34246 35246 32247 33247 34247 35247 32248 33248 34248 35248 32249 33249 34249 35249 32250 33250 34250 35250 32251 33251 34251 35251 32252 33252 34252 35252 32253 33253 34253 35253 32254 33254 34254 35254 32255 33255 34255 35255 32256 33256 34256 35256 32257 33257 34257 35257 32258 33258 34258 35258 32259 33259 34259 35259 32260 33260 34260 35260 32261 33261 34261 35261 32262 33262 34262 35262 32263 33263 34263 35263 32264 33264 34264 35264 32265 33265 34265 35265 32266 33266 34266 35266 32267 33267 34267 35267 32268 33268 34268 35268 32269 33269 34269 35269 32270 33270 34270 35270 32271 33271 34271 35271 32272 33272 34272 35272 32273 33273 34273 35273 32274 33274 34274 35274 32275 33275 34275 35275 32276 33276 34276 35276 32277 33277 34277 35277 32278 33278 34278 35278 32279 33279 34279 35279 32280 33280 34280 35280 32281 33281 34281 35281 32282 33282 34282 35282 32283 33283 34283 35283 32284 33284 34284 35284 32285 33285 34285 35285 32286 33286 34286 35286 32287 33287 34287 35287 32288 33288 34288 35288 32289 33289 34289 35289 32290 33290 34290 35290 32291 33291 34291 35291 32292 33292 34292 35292 32293 33293 34293 35293 32294 33294 34294 35294 32295 33295 34295 35295 32296 33296 34296 35296 32297 33297 34297 35297 32298 33298 34298 35298 32299 33299 34299 35299 32300 33300 34300 35300 32301 33301 34301 35301 32302 33302 34302 35302 32303 33303 34303 35303 32304 33304 34304 35304 32305 33305 34305 35305 32306 33306 34306 35306 32307 33307 34307 35307 32308 33308 34308 35308 32309 33309 34309 35309 32310 33310 34310 35310 32311 33311 34311 35311 32312 33312 34312 35312 32313 33313 34313 35313 32314 33314 34314 35314 32315 33315 34315 35315 32316 33316 34316 35316 32317 33317 34317 35317 32318 33318 34318 35318 32319 33319 34319 35319 32320 33320 34320 35320 32321 33321 34321 35321 32322 33322 34322 35322 32323 33323 34323 35323 32324 33324 34324 35324 32325 33325 34325 35325 32326 33326 34326 35326 32327 33327 34327 35327 32328 33328 34328 35328 32329 33329 34329 35329 32330 33330 34330 35330 32331 33331 34331 35331 32332 33332 34332 35332 32333 33333 34333 35333 32334 33334 34334 35334 32335 33335 34335 35335 32336 33336 34336 35336 32337 33337 34337 35337 32338 33338 34338 35338 32339 33339 34339 35339 32340 33340 34340 35340 32341 33341 34341 35341 32342 33342 34342 35342 32343 33343 34343 35343 32344 33344 34344 35344 32345 33345 34345 35345 32346 33346 34346 35346 32347 33347 34347 35347 32348 33348 34348 35348 32349 33349 34349 35349 32350 33350 34350 35350 32351 33351 34351 35351 32352 33352 34352 35352 32353 33353 34353 35353 32354 33354 34354 35354 32355 33355 34355 35355 32356 33356 34356 35356 32357 33357 34357 35357 32358 33358 34358 35358 32359 33359 34359 35359 32360 33360 34360 35360 32361 33361 34361 35361 32362 33362 34362 35362 32363 33363 34363 35363 32364 33364 34364 35364 32365 33365 34365 35365 32366 33366 34366 35366 32367 33367 34367 35367 32368 33368 34368 35368 32369 33369 34369 35369 32370 33370 34370 35370 32371 33371 34371 35371 32372 33372 34372 35372 32373 33373 34373 35373 32374 33374 34374 35374 32375 33375 34375 35375 32376 33376 34376 35376 32377 33377 34377 35377 32378 33378 34378 35378 32379 33379 34379 35379 32380 33380 34380 35380 32381 33381 34381 35381 32382 33382 34382 35382 32383 33383 34383 35383 32384 33384 34384 35384 32385 33385 34385 35385 32386 33386 34386 35386 32387 33387 34387 35387 32388 33388 34388 35388 32389 33389 34389 35389 32390 33390 34390 35390 32391 33391 34391 35391 32392 33392 34392 35392 32393 33393 34393 35393 32394 33394 34394 35394 32395 33395 34395 35395 32396 33396 34396 35396 32397 33397 34397 35397 32398 33398 34398 35398 32399 33399 34399 35399 32400 33400 34400 35400 32401 33401 34401 35401 32402 33402 34402 35402 32403 33403 34403 35403 32404 33404 34404 35404 32405 33405 34405 35405 32406 33406 34406 35406 32407 33407 34407 35407 32408 33408 34408 35408 32409 33409 34409 35409 32410 33410 34410 35410 32411 33411 34411 35411 32412 33412 34412 35412 32413 33413 34413 35413 32414 33414 34414 35414 32415 33415 34415 35415 32416 33416 34416 35416 32417 33417 34417 35417 32418 33418 34418 35418 32419 33419 34419 35419 32420 33420 34420 35420 32421 33421 34421 35421 32422 33422 34422 35422 32423 33423 34423 35423 32424 33424 34424 35424 32425 33425 34425 35425 32426 33426 34426 35426 32427 33427 34427 35427 32428 33428 34428 35428 32429 33429 34429 35429 32430 33430 34430 35430 32431 33431 34431 35431 32432 33432 34432 35432 32433 33433 34433 35433 32434 33434 34434 35434 32435 33435 34435 35435 32436 33436 34436 35436 32437 33437 34437 35437 32438 33438 34438 35438 32439 33439 34439 35439 32440 33440 34440 35440 32441 33441 34441 35441 32442 33442 34442 35442 32443 33443 34443 35443 32444 33444 34444 35444 32445 33445 34445 35445 32446 33446 34446 35446 32447 33447 34447 35447 32448 33448 34448 35448 32449 33449 34449 35449 32450 33450 34450 35450 32451 33451 34451 35451 32452 33452 34452 35452 32453 33453 34453 35453 32454 33454 34454 35454 32455 33455 34455 35455 32456 33456 34456 35456 32457 33457 34457 35457 32458 33458 34458 35458 32459 33459 34459 35459 32460 33460 34460 35460 32461 33461 34461 35461 32462 33462 34462 35462 32463 33463 34463 35463 32464 33464 34464 35464 32465 33465 34465 35465 32466 33466 34466 35466 32467 33467 34467 35467 32468 33468 34468 35468 32469 33469 34469 35469 32470 33470 34470 35470 32471 33471 34471 35471 32472 33472 34472 35472 32473 33473 34473 35473 32474 33474 34474 35474 32475 33475 34475 35475 32476 33476 34476 35476 32477 33477 34477 35477 32478 33478 34478 35478 32479 33479 34479 35479 32480 33480 34480 35480 32481 33481 34481 35481 32482 33482 34482 35482 32483 33483 34483 35483 32484 33484 34484 35484 32485 33485 34485 35485 32486 33486 34486 35486 32487 33487 34487 35487 32488 33488 34488 35488 32489 33489 34489 35489 32490 33490 34490 35490 32491 33491 34491 35491 32492 33492 34492 35492 32493 33493 34493 35493 32494 33494 34494 35494 32495 33495 34495 35495 32496 33496 34496 35496 32497 33497 34497 35497 32498 33498 34498 35498 32499 33499 34499 35499 32500 33500 34500 35500 32501 33501 34501 35501 32502 33502 34502 35502 32503 33503 34503 35503 32504 33504 34504 35504 32505 33505 34505 35505 32506 33506 34506 35506 32507 33507 34507 35507 32508 33508 34508 35508 32509 33509 34509 35509 32510 33510 34510 35510 32511 33511 34511 35511 32512 33512 34512 35512 32513 33513 34513 35513 32514 33514 34514 35514 32515 33515 34515 35515 32516 33516 34516 35516 32517 33517 34517 35517 32518 33518 34518 35518 32519 33519 34519 35519 32520 33520 34520 35520 32521 33521 34521 35521 32522 33522 34522 35522 32523 33523 34523 35523 32524 33524 34524 35524 32525 33525 34525 35525 32526 33526 34526 35526 32527 33527 34527 35527 32528 33528 34528 35528 32529 33529 34529 35529 32530 33530 34530 35530 32531 33531 34531 35531 32532 33532 34532 35532 32533 33533 34533 35533 32534 33534 34534 35534 32535 33535 34535 35535 32536 33536 34536 35536 32537 33537 34537 35537 32538 33538 34538 35538 32539 33539 34539 35539 32540 33540 34540 35540 32541 33541 34541 35541 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32957 33957 34957 35957 32958 33958 34958 35958 32959 33959 34959 35959 32960 33960 34960 35960 32961 33961 34961 35961 32962 33962 34962 35962 32963 33963 34963 35963 32964 33964 34964 35964 32965 33965 34965 35965 32966 33966 34966 35966 32967 33967 34967 35967 32968 33968 34968 35968 32969 33969 34969 35969 32970 33970 34970 35970 32971 33971 34971 35971 32972 33972 34972 35972 32973 33973 34973 35973 32974 33974 34974 35974 32975 33975 34975 35975 32976 33976 34976 35976 32977 33977 34977 35977 32978 33978 34978 35978 32979 33979 34979 35979 32980 33980 34980 35980 32981 33981 34981 35981 32982 33982 34982 35982 32983 33983 34983 35983 32984 33984 34984 35984 32985 33985 34985 35985 32986 33986 34986 35986 32987 33987 34987 35987 32988 33988 34988 35988 32989 33989 34989 35989 32990 33990 34990 35990 32991 33991 34991 35991 32992 33992 34992 35992 32993 33993 34993 35993 32994 33994 34994 35994 32995 33995 34995 35995 32996 33996 34996 35996 32997 33997 34997 35997 32998 33998 34998 35998 32999 33999 34999 35999 33000 34000 35000 36000 33001 34001 35001 36001 33002 34002 35002 36002

In some embodiments, the sequences that can bind to control materials during detection assay can be selected through the present disclosure. As a non-limiting sequence, the sequences that bind to peanut control material are selected by the present method, which comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 36003 to 37002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to peanut control material may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 37003 to 38002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to peanut control material may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 38003 to 39002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to peanut control material may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 39003 to 40002. In one embodiment, the aptamer of the present disclosure that can be used to detect peanut control material may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 36003 to 40002 listed in Table 11, or variant thereof.

TABLE 11 Aptamer peanut control sequences Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and Sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 36003 37003 38003 39003 36004 37004 38004 39004 36005 37005 38005 39005 36006 37006 38006 39006 36007 37007 38007 39007 36008 37008 38008 39008 36009 37009 38009 39009 36010 37010 38010 39010 36011 37011 38011 39011 36012 37012 38012 39012 36013 37013 38013 39013 36014 37014 38014 39014 36015 37015 38015 39015 36016 37016 38016 39016 36017 37017 38017 39017 36018 37018 38018 39018 36019 37019 38019 39019 36020 37020 38020 39020 36021 37021 38021 39021 36022 37022 38022 39022 36023 37023 38023 39023 36024 37024 38024 39024 36025 37025 38025 39025 36026 37026 38026 39026 36027 37027 38027 39027 36028 37028 38028 39028 36029 37029 38029 39029 36030 37030 38030 39030 36031 37031 38031 39031 36032 37032 38032 39032 36033 37033 38033 39033 36034 37034 38034 39034 36035 37035 38035 39035 36036 37036 38036 39036 36037 37037 38037 39037 36038 37038 38038 39038 36039 37039 38039 39039 36040 37040 38040 39040 36041 37041 38041 39041 36042 37042 38042 39042 36043 37043 38043 39043 36044 37044 38044 39044 36045 37045 38045 39045 36046 37046 38046 39046 36047 37047 38047 39047 36048 37048 38048 39048 36049 37049 38049 39049 36050 37050 38050 39050 36051 37051 38051 39051 36052 37052 38052 39052 36053 37053 38053 39053 36054 37054 38054 39054 36055 37055 38055 39055 36056 37056 38056 39056 36057 37057 38057 39057 36058 37058 38058 39058 36059 37059 38059 39059 36060 37060 38060 39060 36061 37061 38061 39061 36062 37062 38062 39062 36063 37063 38063 39063 36064 37064 38064 39064 36065 37065 38065 39065 36066 37066 38066 39066 36067 37067 38067 39067 36068 37068 38068 39068 36069 37069 38069 39069 36070 37070 38070 39070 36071 37071 38071 39071 36072 37072 38072 39072 36073 37073 38073 39073 36074 37074 38074 39074 36075 37075 38075 39075 36076 37076 38076 39076 36077 37077 38077 39077 36078 37078 38078 39078 36079 37079 38079 39079 36080 37080 38080 39080 36081 37081 38081 39081 36082 37082 38082 39082 36083 37083 38083 39083 36084 37084 38084 39084 36085 37085 38085 39085 36086 37086 38086 39086 36087 37087 38087 39087 36088 37088 38088 39088 36089 37089 38089 39089 36090 37090 38090 39090 36091 37091 38091 39091 36092 37092 38092 39092 36093 37093 38093 39093 36094 37094 38094 39094 36095 37095 38095 39095 36096 37096 38096 39096 36097 37097 38097 39097 36098 37098 38098 39098 36099 37099 38099 39099 36100 37100 38100 39100 36101 37101 38101 39101 36102 37102 38102 39102 36103 37103 38103 39103 36104 37104 38104 39104 36105 37105 38105 39105 36106 37106 38106 39106 36107 37107 38107 39107 36108 37108 38108 39108 36109 37109 38109 39109 36110 37110 38110 39110 36111 37111 38111 39111 36112 37112 38112 39112 36113 37113 38113 39113 36114 37114 38114 39114 36115 37115 38115 39115 36116 37116 38116 39116 36117 37117 38117 39117 36118 37118 38118 39118 36119 37119 38119 39119 36120 37120 38120 39120 36121 37121 38121 39121 36122 37122 38122 39122 36123 37123 38123 39123 36124 37124 38124 39124 36125 37125 38125 39125 36126 37126 38126 39126 36127 37127 38127 39127 36128 37128 38128 39128 36129 37129 38129 39129 36130 37130 38130 39130 36131 37131 38131 39131 36132 37132 38132 39132 36133 37133 38133 39133 36134 37134 38134 39134 36135 37135 38135 39135 36136 37136 38136 39136 36137 37137 38137 39137 36138 37138 38138 39138 36139 37139 38139 39139 36140 37140 38140 39140 36141 37141 38141 39141 36142 37142 38142 39142 36143 37143 38143 39143 36144 37144 38144 39144 36145 37145 38145 39145 36146 37146 38146 39146 36147 37147 38147 39147 36148 37148 38148 39148 36149 37149 38149 39149 36150 37150 38150 39150 36151 37151 38151 39151 36152 37152 38152 39152 36153 37153 38153 39153 36154 37154 38154 39154 36155 37155 38155 39155 36156 37156 38156 39156 36157 37157 38157 39157 36158 37158 38158 39158 36159 37159 38159 39159 36160 37160 38160 39160 36161 37161 38161 39161 36162 37162 38162 39162 36163 37163 38163 39163 36164 37164 38164 39164 36165 37165 38165 39165 36166 37166 38166 39166 36167 37167 38167 39167 36168 37168 38168 39168 36169 37169 38169 39169 36170 37170 38170 39170 36171 37171 38171 39171 36172 37172 38172 39172 36173 37173 38173 39173 36174 37174 38174 39174 36175 37175 38175 39175 36176 37176 38176 39176 36177 37177 38177 39177 36178 37178 38178 39178 36179 37179 38179 39179 36180 37180 38180 39180 36181 37181 38181 39181 36182 37182 38182 39182 36183 37183 38183 39183 36184 37184 38184 39184 36185 37185 38185 39185 36186 37186 38186 39186 36187 37187 38187 39187 36188 37188 38188 39188 36189 37189 38189 39189 36190 37190 38190 39190 36191 37191 38191 39191 36192 37192 38192 39192 36193 37193 38193 39193 36194 37194 38194 39194 36195 37195 38195 39195 36196 37196 38196 39196 36197 37197 38197 39197 36198 37198 38198 39198 36199 37199 38199 39199 36200 37200 38200 39200 36201 37201 38201 39201 36202 37202 38202 39202 36203 37203 38203 39203 36204 37204 38204 39204 36205 37205 38205 39205 36206 37206 38206 39206 36207 37207 38207 39207 36208 37208 38208 39208 36209 37209 38209 39209 36210 37210 38210 39210 36211 37211 38211 39211 36212 37212 38212 39212 36213 37213 38213 39213 36214 37214 38214 39214 36215 37215 38215 39215 36216 37216 38216 39216 36217 37217 38217 39217 36218 37218 38218 39218 36219 37219 38219 39219 36220 37220 38220 39220 36221 37221 38221 39221 36222 37222 38222 39222 36223 37223 38223 39223 36224 37224 38224 39224 36225 37225 38225 39225 36226 37226 38226 39226 36227 37227 38227 39227 36228 37228 38228 39228 36229 37229 38229 39229 36230 37230 38230 39230 36231 37231 38231 39231 36232 37232 38232 39232 36233 37233 38233 39233 36234 37234 38234 39234 36235 37235 38235 39235 36236 37236 38236 39236 36237 37237 38237 39237 36238 37238 38238 39238 36239 37239 38239 39239 36240 37240 38240 39240 36241 37241 38241 39241 36242 37242 38242 39242 36243 37243 38243 39243 36244 37244 38244 39244 36245 37245 38245 39245 36246 37246 38246 39246 36247 37247 38247 39247 36248 37248 38248 39248 36249 37249 38249 39249 36250 37250 38250 39250 36251 37251 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36293 37293 38293 39293 36294 37294 38294 39294 36295 37295 38295 39295 36296 37296 38296 39296 36297 37297 38297 39297 36298 37298 38298 39298 36299 37299 38299 39299 36300 37300 38300 39300 36301 37301 38301 39301 36302 37302 38302 39302 36303 37303 38303 39303 36304 37304 38304 39304 36305 37305 38305 39305 36306 37306 38306 39306 36307 37307 38307 39307 36308 37308 38308 39308 36309 37309 38309 39309 36310 37310 38310 39310 36311 37311 38311 39311 36312 37312 38312 39312 36313 37313 38313 39313 36314 37314 38314 39314 36315 37315 38315 39315 36316 37316 38316 39316 36317 37317 38317 39317 36318 37318 38318 39318 36319 37319 38319 39319 36320 37320 38320 39320 36321 37321 38321 39321 36322 37322 38322 39322 36323 37323 38323 39323 36324 37324 38324 39324 36325 37325 38325 39325 36326 37326 38326 39326 36327 37327 38327 39327 36328 37328 38328 39328 36329 37329 38329 39329 36330 37330 38330 39330 36331 37331 38331 39331 36332 37332 38332 39332 36333 37333 38333 39333 36334 37334 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36459 37459 38459 39459 36460 37460 38460 39460 36461 37461 38461 39461 36462 37462 38462 39462 36463 37463 38463 39463 36464 37464 38464 39464 36465 37465 38465 39465 36466 37466 38466 39466 36467 37467 38467 39467 36468 37468 38468 39468 36469 37469 38469 39469 36470 37470 38470 39470 36471 37471 38471 39471 36472 37472 38472 39472 36473 37473 38473 39473 36474 37474 38474 39474 36475 37475 38475 39475 36476 37476 38476 39476 36477 37477 38477 39477 36478 37478 38478 39478 36479 37479 38479 39479 36480 37480 38480 39480 36481 37481 38481 39481 36482 37482 38482 39482 36483 37483 38483 39483 36484 37484 38484 39484 36485 37485 38485 39485 36486 37486 38486 39486 36487 37487 38487 39487 36488 37488 38488 39488 36489 37489 38489 39489 36490 37490 38490 39490 36491 37491 38491 39491 36492 37492 38492 39492 36493 37493 38493 39493 36494 37494 38494 39494 36495 37495 38495 39495 36496 37496 38496 39496 36497 37497 38497 39497 36498 37498 38498 39498 36499 37499 38499 39499 36500 37500 38500 39500 36501 37501 38501 39501 36502 37502 38502 39502 36503 37503 38503 39503 36504 37504 38504 39504 36505 37505 38505 39505 36506 37506 38506 39506 36507 37507 38507 39507 36508 37508 38508 39508 36509 37509 38509 39509 36510 37510 38510 39510 36511 37511 38511 39511 36512 37512 38512 39512 36513 37513 38513 39513 36514 37514 38514 39514 36515 37515 38515 39515 36516 37516 38516 39516 36517 37517 38517 39517 36518 37518 38518 39518 36519 37519 38519 39519 36520 37520 38520 39520 36521 37521 38521 39521 36522 37522 38522 39522 36523 37523 38523 39523 36524 37524 38524 39524 36525 37525 38525 39525 36526 37526 38526 39526 36527 37527 38527 39527 36528 37528 38528 39528 36529 37529 38529 39529 36530 37530 38530 39530 36531 37531 38531 39531 36532 37532 38532 39532 36533 37533 38533 39533 36534 37534 38534 39534 36535 37535 38535 39535 36536 37536 38536 39536 36537 37537 38537 39537 36538 37538 38538 39538 36539 37539 38539 39539 36540 37540 38540 39540 36541 37541 38541 39541 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36791 37791 38791 39791 36792 37792 38792 39792 36793 37793 38793 39793 36794 37794 38794 39794 36795 37795 38795 39795 36796 37796 38796 39796 36797 37797 38797 39797 36798 37798 38798 39798 36799 37799 38799 39799 36800 37800 38800 39800 36801 37801 38801 39801 36802 37802 38802 39802 36803 37803 38803 39803 36804 37804 38804 39804 36805 37805 38805 39805 36806 37806 38806 39806 36807 37807 38807 39807 36808 37808 38808 39808 36809 37809 38809 39809 36810 37810 38810 39810 36811 37811 38811 39811 36812 37812 38812 39812 36813 37813 38813 39813 36814 37814 38814 39814 36815 37815 38815 39815 36816 37816 38816 39816 36817 37817 38817 39817 36818 37818 38818 39818 36819 37819 38819 39819 36820 37820 38820 39820 36821 37821 38821 39821 36822 37822 38822 39822 36823 37823 38823 39823 36824 37824 38824 39824 36825 37825 38825 39825 36826 37826 38826 39826 36827 37827 38827 39827 36828 37828 38828 39828 36829 37829 38829 39829 36830 37830 38830 39830 36831 37831 38831 39831 36832 37832 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36874 37874 38874 39874 36875 37875 38875 39875 36876 37876 38876 39876 36877 37877 38877 39877 36878 37878 38878 39878 36879 37879 38879 39879 36880 37880 38880 39880 36881 37881 38881 39881 36882 37882 38882 39882 36883 37883 38883 39883 36884 37884 38884 39884 36885 37885 38885 39885 36886 37886 38886 39886 36887 37887 38887 39887 36888 37888 38888 39888 36889 37889 38889 39889 36890 37890 38890 39890 36891 37891 38891 39891 36892 37892 38892 39892 36893 37893 38893 39893 36894 37894 38894 39894 36895 37895 38895 39895 36896 37896 38896 39896 36897 37897 38897 39897 36898 37898 38898 39898 36899 37899 38899 39899 36900 37900 38900 39900 36901 37901 38901 39901 36902 37902 38902 39902 36903 37903 38903 39903 36904 37904 38904 39904 36905 37905 38905 39905 36906 37906 38906 39906 36907 37907 38907 39907 36908 37908 38908 39908 36909 37909 38909 39909 36910 37910 38910 39910 36911 37911 38911 39911 36912 37912 38912 39912 36913 37913 38913 39913 36914 37914 38914 39914 36915 37915 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36957 37957 38957 39957 36958 37958 38958 39958 36959 37959 38959 39959 36960 37960 38960 39960 36961 37961 38961 39961 36962 37962 38962 39962 36963 37963 38963 39963 36964 37964 38964 39964 36965 37965 38965 39965 36966 37966 38966 39966 36967 37967 38967 39967 36968 37968 38968 39968 36969 37969 38969 39969 36970 37970 38970 39970 36971 37971 38971 39971 36972 37972 38972 39972 36973 37973 38973 39973 36974 37974 38974 39974 36975 37975 38975 39975 36976 37976 38976 39976 36977 37977 38977 39977 36978 37978 38978 39978 36979 37979 38979 39979 36980 37980 38980 39980 36981 37981 38981 39981 36982 37982 38982 39982 36983 37983 38983 39983 36984 37984 38984 39984 36985 37985 38985 39985 36986 37986 38986 39986 36987 37987 38987 39987 36988 37988 38988 39988 36989 37989 38989 39989 36990 37990 38990 39990 36991 37991 38991 39991 36992 37992 38992 39992 36993 37993 38993 39993 36994 37994 38994 39994 36995 37995 38995 39995 36996 37996 38996 39996 36997 37997 38997 39997 36998 37998 38998 39998 36999 37999 38999 39999 37000 38000 39000 40000 37001 38001 39001 40001 37002 38002 39002 40002

In some embodiments, the sequences that specifically bind to gluten comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 40003 to 41002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to gluten may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 41003 to 42002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to gluten may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 42003 to 43002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to gluten may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 43003 to 44002. In one embodiment, the aptamer of the present disclosure that specifically binds to gluten may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 40003 to 44002 listed in Table 12, or variant thereof.

TABLE 12 Aptamer sequences against gluten Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and Sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 40003 41003 42003 43003 40004 41004 42004 43004 40005 41005 42005 43005 40006 41006 42006 43006 40007 41007 42007 43007 40008 41008 42008 43008 40009 41009 42009 43009 40010 41010 42010 43010 40011 41011 42011 43011 40012 41012 42012 43012 40013 41013 42013 43013 40014 41014 42014 43014 40015 41015 42015 43015 40016 41016 42016 43016 40017 41017 42017 43017 40018 41018 42018 43018 40019 41019 42019 43019 40020 41020 42020 43020 40021 41021 42021 43021 40022 41022 42022 43022 40023 41023 42023 43023 40024 41024 42024 43024 40025 41025 42025 43025 40026 41026 42026 43026 40027 41027 42027 43027 40028 41028 42028 43028 40029 41029 42029 43029 40030 41030 42030 43030 40031 41031 42031 43031 40032 41032 42032 43032 40033 41033 42033 43033 40034 41034 42034 43034 40035 41035 42035 43035 40036 41036 42036 43036 40037 41037 42037 43037 40038 41038 42038 43038 40039 41039 42039 43039 40040 41040 42040 43040 40041 41041 42041 43041 40042 41042 42042 43042 40043 41043 42043 43043 40044 41044 42044 43044 40045 41045 42045 43045 40046 41046 42046 43046 40047 41047 42047 43047 40048 41048 42048 43048 40049 41049 42049 43049 40050 41050 42050 43050 40051 41051 42051 43051 40052 41052 42052 43052 40053 41053 42053 43053 40054 41054 42054 43054 40055 41055 42055 43055 40056 41056 42056 43056 40057 41057 42057 43057 40058 41058 42058 43058 40059 41059 42059 43059 40060 41060 42060 43060 40061 41061 42061 43061 40062 41062 42062 43062 40063 41063 42063 43063 40064 41064 42064 43064 40065 41065 42065 43065 40066 41066 42066 43066 40067 41067 42067 43067 40068 41068 42068 43068 40069 41069 42069 43069 40070 41070 42070 43070 40071 41071 42071 43071 40072 41072 42072 43072 40073 41073 42073 43073 40074 41074 42074 43074 40075 41075 42075 43075 40076 41076 42076 43076 40077 41077 42077 43077 40078 41078 42078 43078 40079 41079 42079 43079 40080 41080 42080 43080 40081 41081 42081 43081 40082 41082 42082 43082 40083 41083 42083 43083 40084 41084 42084 43084 40085 41085 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40127 41127 42127 43127 40128 41128 42128 43128 40129 41129 42129 43129 40130 41130 42130 43130 40131 41131 42131 43131 40132 41132 42132 43132 40133 41133 42133 43133 40134 41134 42134 43134 40135 41135 42135 43135 40136 41136 42136 43136 40137 41137 42137 43137 40138 41138 42138 43138 40139 41139 42139 43139 40140 41140 42140 43140 40141 41141 42141 43141 40142 41142 42142 43142 40143 41143 42143 43143 40144 41144 42144 43144 40145 41145 42145 43145 40146 41146 42146 43146 40147 41147 42147 43147 40148 41148 42148 43148 40149 41149 42149 43149 40150 41150 42150 43150 40151 41151 42151 43151 40152 41152 42152 43152 40153 41153 42153 43153 40154 41154 42154 43154 40155 41155 42155 43155 40156 41156 42156 43156 40157 41157 42157 43157 40158 41158 42158 43158 40159 41159 42159 43159 40160 41160 42160 43160 40161 41161 42161 43161 40162 41162 42162 43162 40163 41163 42163 43163 40164 41164 42164 43164 40165 41165 42165 43165 40166 41166 42166 43166 40167 41167 42167 43167 40168 41168 42168 43168 40169 41169 42169 43169 40170 41170 42170 43170 40171 41171 42171 43171 40172 41172 42172 43172 40173 41173 42173 43173 40174 41174 42174 43174 40175 41175 42175 43175 40176 41176 42176 43176 40177 41177 42177 43177 40178 41178 42178 43178 40179 41179 42179 43179 40180 41180 42180 43180 40181 41181 42181 43181 40182 41182 42182 43182 40183 41183 42183 43183 40184 41184 42184 43184 40185 41185 42185 43185 40186 41186 42186 43186 40187 41187 42187 43187 40188 41188 42188 43188 40189 41189 42189 43189 40190 41190 42190 43190 40191 41191 42191 43191 40192 41192 42192 43192 40193 41193 42193 43193 40194 41194 42194 43194 40195 41195 42195 43195 40196 41196 42196 43196 40197 41197 42197 43197 40198 41198 42198 43198 40199 41199 42199 43199 40200 41200 42200 43200 40201 41201 42201 43201 40202 41202 42202 43202 40203 41203 42203 43203 40204 41204 42204 43204 40205 41205 42205 43205 40206 41206 42206 43206 40207 41207 42207 43207 40208 41208 42208 43208 40209 41209 42209 43209 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40957 41957 42957 43957 40958 41958 42958 43958 40959 41959 42959 43959 40960 41960 42960 43960 40961 41961 42961 43961 40962 41962 42962 43962 40963 41963 42963 43963 40964 41964 42964 43964 40965 41965 42965 43965 40966 41966 42966 43966 40967 41967 42967 43967 40968 41968 42968 43968 40969 41969 42969 43969 40970 41970 42970 43970 40971 41971 42971 43971 40972 41972 42972 43972 40973 41973 42973 43973 40974 41974 42974 43974 40975 41975 42975 43975 40976 41976 42976 43976 40977 41977 42977 43977 40978 41978 42978 43978 40979 41979 42979 43979 40980 41980 42980 43980 40981 41981 42981 43981 40982 41982 42982 43982 40983 41983 42983 43983 40984 41984 42984 43984 40985 41985 42985 43985 40986 41986 42986 43986 40987 41987 42987 43987 40988 41988 42988 43988 40989 41989 42989 43989 40990 41990 42990 43990 40991 41991 42991 43991 40992 41992 42992 43992 40993 41993 42993 43993 40994 41994 42994 43994 40995 41995 42995 43995 40996 41996 42996 43996 40997 41997 42997 43997 40998 41998 42998 43998 40999 41999 42999 43999 41000 42000 43000 44000 41001 42001 43001 44001 41002 42002 43002 44002

In some embodiments, the sequences that specifically bind to whey comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 44003 to 45002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to whey may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 45003 to 46002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to whey may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 46003 to 47002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to whey may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 47003 to 48002. In one embodiment, the aptamer of the present disclosure that specifically binds to whey may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 44003 to 48002 listed in Table 13, or variant thereof.

TABLE 13 Aptamer sequences against whey Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 44003 45003 46003 47003 44004 45004 46004 47004 44005 45005 46005 47005 44006 45006 46006 47006 44007 45007 46007 47007 44008 45008 46008 47008 44009 45009 46009 47009 44010 45010 46010 47010 44011 45011 46011 47011 44012 45012 46012 47012 44013 45013 46013 47013 44014 45014 46014 47014 44015 45015 46015 47015 44016 45016 46016 47016 44017 45017 46017 47017 44018 45018 46018 47018 44019 45019 46019 47019 44020 45020 46020 47020 44021 45021 46021 47021 44022 45022 46022 47022 44023 45023 46023 47023 44024 45024 46024 47024 44025 45025 46025 47025 44026 45026 46026 47026 44027 45027 46027 47027 44028 45028 46028 47028 44029 45029 46029 47029 44030 45030 46030 47030 44031 45031 46031 47031 44032 45032 46032 47032 44033 45033 46033 47033 44034 45034 46034 47034 44035 45035 46035 47035 44036 45036 46036 47036 44037 45037 46037 47037 44038 45038 46038 47038 44039 45039 46039 47039 44040 45040 46040 47040 44041 45041 46041 47041 44042 45042 46042 47042 44043 45043 46043 47043 44044 45044 46044 47044 44045 45045 46045 47045 44046 45046 46046 47046 44047 45047 46047 47047 44048 45048 46048 47048 44049 45049 46049 47049 44050 45050 46050 47050 44051 45051 46051 47051 44052 45052 46052 47052 44053 45053 46053 47053 44054 45054 46054 47054 44055 45055 46055 47055 44056 45056 46056 47056 44057 45057 46057 47057 44058 45058 46058 47058 44059 45059 46059 47059 44060 45060 46060 47060 44061 45061 46061 47061 44062 45062 46062 47062 44063 45063 46063 47063 44064 45064 46064 47064 44065 45065 46065 47065 44066 45066 46066 47066 44067 45067 46067 47067 44068 45068 46068 47068 44069 45069 46069 47069 44070 45070 46070 47070 44071 45071 46071 47071 44072 45072 46072 47072 44073 45073 46073 47073 44074 45074 46074 47074 44075 45075 46075 47075 44076 45076 46076 47076 44077 45077 46077 47077 44078 45078 46078 47078 44079 45079 46079 47079 44080 45080 46080 47080 44081 45081 46081 47081 44082 45082 46082 47082 44083 45083 46083 47083 44084 45084 46084 47084 44085 45085 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44127 45127 46127 47127 44128 45128 46128 47128 44129 45129 46129 47129 44130 45130 46130 47130 44131 45131 46131 47131 44132 45132 46132 47132 44133 45133 46133 47133 44134 45134 46134 47134 44135 45135 46135 47135 44136 45136 46136 47136 44137 45137 46137 47137 44138 45138 46138 47138 44139 45139 46139 47139 44140 45140 46140 47140 44141 45141 46141 47141 44142 45142 46142 47142 44143 45143 46143 47143 44144 45144 46144 47144 44145 45145 46145 47145 44146 45146 46146 47146 44147 45147 46147 47147 44148 45148 46148 47148 44149 45149 46149 47149 44150 45150 46150 47150 44151 45151 46151 47151 44152 45152 46152 47152 44153 45153 46153 47153 44154 45154 46154 47154 44155 45155 46155 47155 44156 45156 46156 47156 44157 45157 46157 47157 44158 45158 46158 47158 44159 45159 46159 47159 44160 45160 46160 47160 44161 45161 46161 47161 44162 45162 46162 47162 44163 45163 46163 47163 44164 45164 46164 47164 44165 45165 46165 47165 44166 45166 46166 47166 44167 45167 46167 47167 44168 45168 46168 47168 44169 45169 46169 47169 44170 45170 46170 47170 44171 45171 46171 47171 44172 45172 46172 47172 44173 45173 46173 47173 44174 45174 46174 47174 44175 45175 46175 47175 44176 45176 46176 47176 44177 45177 46177 47177 44178 45178 46178 47178 44179 45179 46179 47179 44180 45180 46180 47180 44181 45181 46181 47181 44182 45182 46182 47182 44183 45183 46183 47183 44184 45184 46184 47184 44185 45185 46185 47185 44186 45186 46186 47186 44187 45187 46187 47187 44188 45188 46188 47188 44189 45189 46189 47189 44190 45190 46190 47190 44191 45191 46191 47191 44192 45192 46192 47192 44193 45193 46193 47193 44194 45194 46194 47194 44195 45195 46195 47195 44196 45196 46196 47196 44197 45197 46197 47197 44198 45198 46198 47198 44199 45199 46199 47199 44200 45200 46200 47200 44201 45201 46201 47201 44202 45202 46202 47202 44203 45203 46203 47203 44204 45204 46204 47204 44205 45205 46205 47205 44206 45206 46206 47206 44207 45207 46207 47207 44208 45208 46208 47208 44209 45209 46209 47209 44210 45210 46210 47210 44211 45211 46211 47211 44212 45212 46212 47212 44213 45213 46213 47213 44214 45214 46214 47214 44215 45215 46215 47215 44216 45216 46216 47216 44217 45217 46217 47217 44218 45218 46218 47218 44219 45219 46219 47219 44220 45220 46220 47220 44221 45221 46221 47221 44222 45222 46222 47222 44223 45223 46223 47223 44224 45224 46224 47224 44225 45225 46225 47225 44226 45226 46226 47226 44227 45227 46227 47227 44228 45228 46228 47228 44229 45229 46229 47229 44230 45230 46230 47230 44231 45231 46231 47231 44232 45232 46232 47232 44233 45233 46233 47233 44234 45234 46234 47234 44235 45235 46235 47235 44236 45236 46236 47236 44237 45237 46237 47237 44238 45238 46238 47238 44239 45239 46239 47239 44240 45240 46240 47240 44241 45241 46241 47241 44242 45242 46242 47242 44243 45243 46243 47243 44244 45244 46244 47244 44245 45245 46245 47245 44246 45246 46246 47246 44247 45247 46247 47247 44248 45248 46248 47248 44249 45249 46249 47249 44250 45250 46250 47250 44251 45251 46251 47251 44252 45252 46252 47252 44253 45253 46253 47253 44254 45254 46254 47254 44255 45255 46255 47255 44256 45256 46256 47256 44257 45257 46257 47257 44258 45258 46258 47258 44259 45259 46259 47259 44260 45260 46260 47260 44261 45261 46261 47261 44262 45262 46262 47262 44263 45263 46263 47263 44264 45264 46264 47264 44265 45265 46265 47265 44266 45266 46266 47266 44267 45267 46267 47267 44268 45268 46268 47268 44269 45269 46269 47269 44270 45270 46270 47270 44271 45271 46271 47271 44272 45272 46272 47272 44273 45273 46273 47273 44274 45274 46274 47274 44275 45275 46275 47275 44276 45276 46276 47276 44277 45277 46277 47277 44278 45278 46278 47278 44279 45279 46279 47279 44280 45280 46280 47280 44281 45281 46281 47281 44282 45282 46282 47282 44283 45283 46283 47283 44284 45284 46284 47284 44285 45285 46285 47285 44286 45286 46286 47286 44287 45287 46287 47287 44288 45288 46288 47288 44289 45289 46289 47289 44290 45290 46290 47290 44291 45291 46291 47291 44292 45292 46292 47292 44293 45293 46293 47293 44294 45294 46294 47294 44295 45295 46295 47295 44296 45296 46296 47296 44297 45297 46297 47297 44298 45298 46298 47298 44299 45299 46299 47299 44300 45300 46300 47300 44301 45301 46301 47301 44302 45302 46302 47302 44303 45303 46303 47303 44304 45304 46304 47304 44305 45305 46305 47305 44306 45306 46306 47306 44307 45307 46307 47307 44308 45308 46308 47308 44309 45309 46309 47309 44310 45310 46310 47310 44311 45311 46311 47311 44312 45312 46312 47312 44313 45313 46313 47313 44314 45314 46314 47314 44315 45315 46315 47315 44316 45316 46316 47316 44317 45317 46317 47317 44318 45318 46318 47318 44319 45319 46319 47319 44320 45320 46320 47320 44321 45321 46321 47321 44322 45322 46322 47322 44323 45323 46323 47323 44324 45324 46324 47324 44325 45325 46325 47325 44326 45326 46326 47326 44327 45327 46327 47327 44328 45328 46328 47328 44329 45329 46329 47329 44330 45330 46330 47330 44331 45331 46331 47331 44332 45332 46332 47332 44333 45333 46333 47333 44334 45334 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46998 47998 44999 45999 46999 47999 45000 46000 47000 48000 45001 46001 47001 48001 45002 46002 47002 48002

In some embodiments, the sequences that specifically bind to casein comprise an inner sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs. 48003 to 49002. In some examples, a short nucleic acid sequence may be affixed to the 5-end of the inner sequence. The short nucleic acid sequence may be the 5′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 1. Accordingly, the aptamer that specifically binds to casein may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 49003 to 50002. In other examples, a short nucleic acid sequence may be affixed to the 3-end of the inner sequence. The short nucleic acid sequence may be the 3′ primer sequence used in the random ssDNA library, i.e. the nucleic acid sequence of SEQ ID NO. 2. Accordingly, the aptamer that specifically binds to casein may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 50003 to 51002. In other examples, the inner sequence may comprise a 5′ end short sequence (i.e., SEQ ID NO. 1) and a 3′ end short sequence (i.e., SEQ ID NO. 2). Accordingly, the aptamer that specifically binds to casein may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51003 to 52002. In one embodiment, the aptamer of the present disclosure that specifically binds to casein may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 48003 to 52002 listed in Table 14, or variant thereof.

TABLE 14 Aptamer sequences against casein Aptamer sequence 5′-sequence 5′-sequence Inner and inner Inner and inner sequence and sequence and sequence sequence 3′-sequence 3′-sequence (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) (SEQ ID NO.) 48003 49003 50003 51003 48004 49004 50004 51004 48005 49005 50005 51005 48006 49006 50006 51006 48007 49007 50007 51007 48008 49008 50008 51008 48009 49009 50009 51009 48010 49010 50010 51010 48011 49011 50011 51011 48012 49012 50012 51012 48013 49013 50013 51013 48014 49014 50014 51014 48015 49015 50015 51015 48016 49016 50016 51016 48017 49017 50017 51017 48018 49018 50018 51018 48019 49019 50019 51019 48020 49020 50020 51020 48021 49021 50021 51021 48022 49022 50022 51022 48023 49023 50023 51023 48024 49024 50024 51024 48025 49025 50025 51025 48026 49026 50026 51026 48027 49027 50027 51027 48028 49028 50028 51028 48029 49029 50029 51029 48030 49030 50030 51030 48031 49031 50031 51031 48032 49032 50032 51032 48033 49033 50033 51033 48034 49034 50034 51034 48035 49035 50035 51035 48036 49036 50036 51036 48037 49037 50037 51037 48038 49038 50038 51038 48039 49039 50039 51039 48040 49040 50040 51040 48041 49041 50041 51041 48042 49042 50042 51042 48043 49043 50043 51043 48044 49044 50044 51044 48045 49045 50045 51045 48046 49046 50046 51046 48047 49047 50047 51047 48048 49048 50048 51048 48049 49049 50049 51049 48050 49050 50050 51050 48051 49051 50051 51051 48052 49052 50052 51052 48053 49053 50053 51053 48054 49054 50054 51054 48055 49055 50055 51055 48056 49056 50056 51056 48057 49057 50057 51057 48058 49058 50058 51058 48059 49059 50059 51059 48060 49060 50060 51060 48061 49061 50061 51061 48062 49062 50062 51062 48063 49063 50063 51063 48064 49064 50064 51064 48065 49065 50065 51065 48066 49066 50066 51066 48067 49067 50067 51067 48068 49068 50068 51068 48069 49069 50069 51069 48070 49070 50070 51070 48071 49071 50071 51071 48072 49072 50072 51072 48073 49073 50073 51073 48074 49074 50074 51074 48075 49075 50075 51075 48076 49076 50076 51076 48077 49077 50077 51077 48078 49078 50078 51078 48079 49079 50079 51079 48080 49080 50080 51080 48081 49081 50081 51081 48082 49082 50082 51082 48083 49083 50083 51083 48084 49084 50084 51084 48085 49085 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48127 49127 50127 51127 48128 49128 50128 51128 48129 49129 50129 51129 48130 49130 50130 51130 48131 49131 50131 51131 48132 49132 50132 51132 48133 49133 50133 51133 48134 49134 50134 51134 48135 49135 50135 51135 48136 49136 50136 51136 48137 49137 50137 51137 48138 49138 50138 51138 48139 49139 50139 51139 48140 49140 50140 51140 48141 49141 50141 51141 48142 49142 50142 51142 48143 49143 50143 51143 48144 49144 50144 51144 48145 49145 50145 51145 48146 49146 50146 51146 48147 49147 50147 51147 48148 49148 50148 51148 48149 49149 50149 51149 48150 49150 50150 51150 48151 49151 50151 51151 48152 49152 50152 51152 48153 49153 50153 51153 48154 49154 50154 51154 48155 49155 50155 51155 48156 49156 50156 51156 48157 49157 50157 51157 48158 49158 50158 51158 48159 49159 50159 51159 48160 49160 50160 51160 48161 49161 50161 51161 48162 49162 50162 51162 48163 49163 50163 51163 48164 49164 50164 51164 48165 49165 50165 51165 48166 49166 50166 51166 48167 49167 50167 51167 48168 49168 50168 51168 48169 49169 50169 51169 48170 49170 50170 51170 48171 49171 50171 51171 48172 49172 50172 51172 48173 49173 50173 51173 48174 49174 50174 51174 48175 49175 50175 51175 48176 49176 50176 51176 48177 49177 50177 51177 48178 49178 50178 51178 48179 49179 50179 51179 48180 49180 50180 51180 48181 49181 50181 51181 48182 49182 50182 51182 48183 49183 50183 51183 48184 49184 50184 51184 48185 49185 50185 51185 48186 49186 50186 51186 48187 49187 50187 51187 48188 49188 50188 51188 48189 49189 50189 51189 48190 49190 50190 51190 48191 49191 50191 51191 48192 49192 50192 51192 48193 49193 50193 51193 48194 49194 50194 51194 48195 49195 50195 51195 48196 49196 50196 51196 48197 49197 50197 51197 48198 49198 50198 51198 48199 49199 50199 51199 48200 49200 50200 51200 48201 49201 50201 51201 48202 49202 50202 51202 48203 49203 50203 51203 48204 49204 50204 51204 48205 49205 50205 51205 48206 49206 50206 51206 48207 49207 50207 51207 48208 49208 50208 51208 48209 49209 50209 51209 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In some embodiments, the SPN of the present disclosure comprises an aptamer selected by the present method and a short oligonucleotide anchor sequence that may be coated to a solid support. The short anchor oligonucleotide comprises a nucleic acid sequence complementary to a portion of the same aptamer sequence. In some embodiments, a SPN for detecting peanut allergen comprises an aptamer sequence selected from the group consisting of SEQ ID Nos. 3 to 4002 and one or more short anchor sequences that are complementary to the aptamer sequence. As a non-limiting example, the complementary sequence may comprise a nucleic acid sequence selected from SEQ ID NOs. 52003 to 52042 (as shown in Table 15). In some embodiments, the anchor oligonucleotide may be modified to contain a spacer at one end of the sequence. As a non-limiting example, the anchor sequence is modified to contain either a 12-Carbon atom spacer or a 6-Carbon atom spacer at the 5′ end of the sequence (Table 15), or a polyA tail at one end of the sequence. The short complementary sequences may be covalently attached to the solid support (e.g., a glass or plastic chip) directly or through a linker. Accordingly, the length of the linker (carbon atoms or polyA tail) from the solid surface can prevent steric hindrance and reduce the probability of interference due to auto-fluorescence of matrices.

TABLE 15 Short complementary anchor sequences SEQ ID NO. Sequence (5′-3′) 52003 6C-AAAAATCAAGTGGTC 52004 12C-AAAAATCAAGTGGTC 52005 6C-AAAAATCAAGTG 52006 12C-AAAAATCAAGTG 52007 6C-AAAAAAGTGGTC 52008 12C-AAAAAAGTGGTC 52009 6C-AAAAATCAAGAGGTC 52010 12C-AAAAATCAAGAGGTC 52011 6C-AAAAATCAACAGGTC 52012 12C-AAAAATCAACAGGTC 52013 6C-AAAAAAGTGGTCATG 52014 12C-AAAAAAGTGGTCATG 52015 6C-AAAAATGGTCATGTA 52016 12C-AAAAATGGTCATGTA 52017 6C-AAAAATGGTCAT 52018 12C-AAAAATGGTCAT 52019 6C-AAAAATCATGTA 52020 12C-AAAAATCATGTA 52021 6C-AAAAATGGTCTTGTA 52022 12C-AAAAATGGTCTTGTA 52023 6C-AAAAATGGTGTTGTA 52024 12C-AAAAATGGTGTTGTA 52025 6C-AAAAATCATGTACTA 52026 12C-AAAAATCATGTACTA 52027 6C-AAAAACTCTTCCCTA 52028 12C-AAAAACTCTTCCCTA 52029 6C-AAAAACTCTTCC 52030 12C-AAAAACTCTTCC 52031 6C-AAAAATTCCCTA 52032 12C-AAAAATTCCCTA 52033 6C-AAAAACTCTTGCCTA 52034 12C-AAAAACTCTTGCCTA 52035 6C-AAAAACTCTAGCCTA 52036 12C-AAAAACTCTAGCCTA 52037 6C-AAAAAGATCAGGCCA 52038 6C-AAAAACACTTGCGGT 52039 6C-AAAAACACGGACACG 52040 6C-AAAAAGGCCATGCTT 52041 6C-AAAAAGCTGCTCATC 52042 6C-AAAAAGAAGACACAC

Detection Kits

In some embodiments, the present disclosure provides a detection kit for allergen detection. The kit comprises (a) a SPN comprising an aptamer sequence that specifically binds to a target of interest, wherein the aptamer does not bind to its complementary sequence in the presence of the target of interest; and (b) a solid support of which the surface is coated with short nucleic acid sequences that are complementary to the sequence of the aptamer. The detection kit may further comprise one or more buffer solutions and other reagents. The buffers are suitable for preparing sample solutions, SPN solutions, and/or other solutions necessary for running a detection assay (e.g., wash buffers). One or more of these kit components may be separated into individual containers, or they may be provided in their aggregated state. In some embodiments, the kit may comprise multiple SPNs specific to multiple allergen targets. For example, the kit may comprise a panel of SPNs specific to peanut and common tree nuts including almond, brazil nut, cashew, hazel nut, pecan, pistachio and walnut.

In some embodiments, the detection kit may further comprise one or more control aptamer sequences; the control sequences may be used to measure total protein and normalize the baseline. For example, a detection kit comprising SPNs specific to peanut for peanut detection may comprise peanut control sequences that can measure total protein and normalize the baseline during peanut detection. As a non-limiting example, a peanut detection kit may comprise one or more peanut specific aptamers comprising nucleic acid sequences selected from SEQ ID NOs. 3-4002 and one or more peanut control aptamers comprising nucleic acid sequences selected from SEQ ID NOs. 36003 to 40002.

Detection Assays

In some embodiments, the present disclosure provides a method for detecting the presence and/or absence of an allergen in a food sample, the method comprising the steps of (i) preparing a sample to be tested solution and a SPN solution; (ii) mixing the sample and SPN solutions and incubating the mixture to induce the binding of the target to the SPN; (iii) contacting the mixture to a solid support that is coated with short oligonucleotides comprising sequences complementary to the SPN; and (iv) measuring a signal and detecting the presence and/or absence of the allergen of interest. The SPN may be labeled with a fluorophore at one end of the sequence, e.g., Cy5 and Alexa Fluor 647.

In some embodiments, the solid support is a glass chip (e.g., a borosilicate glass chip) wherein the surface of the glass chip is divided into several panels including at least one reactive panel and at least two control panels. The reactive panel of the glass chip are covalently coated with short oligonucleotides comprising sequences complementary to the SPN to which the SPN can hybridize to form a double stranded nucleic acid when the SPN is free from the binding of the target of interest. The reactive panel may be flanked by two control panels at each side. The control panels may be coated with random sequences that do not bind to the SPN nor the target.

The chip can be any size suitable for the use in a detection device/system, e.g., 10×10 mm. In some embodiments, the detection chip may be a plastic chip.

In some embodiments, the food sample may be processed with a homogenization buffer that contains a SPN specific to an allergen of interest (e.g., peanut). The food slurry passes over a reactive panel on a glass chip, embedded in a cartridge designed to position the chip to face a laser and an optical sensor. Wash buffer is flowed over the reactive panel, thereby removing any non-specific binding interactions from the panel. Multiple steps of the assay are read by the optical sensor and analyzed by an algorithm to provide an “allergen detected” or “allergen not detected” response. In the absence of the target allergen, the SPN is free to bind to the complementary oligonucleotides on the reactive panel, resulting in a high fluorescence signal. In the presence of the target allergen, the SPN:complement binding interface is occluded, thereby resulting in a decrease in fluorescence signal on the reactive panel.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

EXAMPLES Example 1: Positive Graphene Oxide (GO)-SELEX Selection

As illustrated in FIG. 1, to begin a round of SELEX, the ssDNA molecules from either a random DNA library (round 1) or from the previous round (enriched library) is diluted at a concentration in water (e.g., 20 ng/μL). A target protein solution is prepared in the appropriate extraction buffer and diluted to a desired concentration depending on the round. A volume of the diluted ssDNA molecules solution (e.g., 100 μL) and a volume of the target protein solution (e.g., 300 μL) is mixed and the resulting mixture is incubated at room temperature with shaking for a set of time depending on the round. A graphene oxide (GO) solution diluted to a defined amount in the extraction buffer (e.g., 600 μL) is added to the ssDNA molecules and target mixture. Graphene oxide (GO) can adsorb the unbound sequences and let the sequences bound to the target free. The unbound sequences and GO are then removed by centrifugation. The ssDNA/target/GO mixture is incubated for 20 minutes at room temperature with shaking, during which any ssDNA that is not bound to the target material will be adsorbed onto the GO surface. After 20 minutes, the mixture is centrifuged at 10,000 g for 3 minutes, and the supernatant, containing ssDNA bound to the target protein and excess target protein, is collected. The pellet containing the GO and ssDNA adsorbed onto the GO surface is discarded.

To separate the bound ssDNAs from the target, 10% Strataclean resin is added to the collected supernatant which contains target protein and ssDNA complexes. The resulting mixture is heated to 80° C. for 3 minutes, followed by centrifugation at 10,000 g for 3 minutes. The pellet containing the resin bound target proteins is discarded and the supernatant is collected. The strataclean step is repeated for at least one more round. The concentration of ssDNAs in the final supernatant is measured and compared to the initial concentration prior to addition of target proteins and GO. The ssDNA ratio after each round of selection is used to determine if further round selection is necessary. For example, if the ratio is below 50%, the same conditions are repeated in the next round until recovery improves.

The collected final ssDNA pool is then amplified by PCR using a biotinylated reverse primer and a Cy5-tagged forward primer. The PCR amplified DNAs are cleaned for removal of any residual reagent (e.g., PCR Clean Up Kit) and measured for the concentration of DNA molecules. The clean PCR product is added to streptavidin coated magnetic beads. The biotinylated complimentary strand binds the streptavidin coated beads, then base is added to denature the dsDNA molecules. Using a magnet, the beads, with the biotinylated complimentary strand still bound, are pulled out of solution and the desired ssDNA strands with the Cy5-tag are collected. The isolated ssDNA pool is concentrated, measured and prepared for next selection round.

Example 2: Positive On-Glass Selection

The ssDNA pool from Example 1 is diluted to 0.2 ng/μL in the extraction buffer. The same target solution is prepared and diluted to stringent conditions. 50 μL of the ssDNA solution is mixed with 50 μL of target protein and incubated for 1 minute at room temperature with shaking. This ssDNA/protein mixture is then added to two wells of a 16-well slide containing short complimentary anchors to the primer regions of the ssDNA molecules. After incubation for 1 minute at room temperature with shaking, the ssDNA/protein mixture is transferred to the next two wells of the same slide. This process is repeated for a total of eight incubations. Following the final incubation, the ssDNA/protein mixture is collected. The cleaning, amplification, and strand separation steps are the same as in the positive GO-SELEX selection (See Example 1). This on-glass selection can be repeated multiple times until the recovery ratio is acceptable.

Example 3: Non-Binding On-Glass Counter Selection

The ssDNA molecules from the final round of positive on-glass SELEX (Example 2) are diluted to a concentration of 0.1 ng/μL in extraction buffer. 50 μL of the ssDNA solution is added to two wells of a new 16-well slide as described above. Without any protein present, all sequences in the pool that are capable of binding the complimentary sequences should bind. After incubation for 1 minute at room temperature with shaking, the ssDNA solution is transferred to the next two wells of the same slide and incubated for another 1 minute. This process is repeated for a total of eight incubations. Following the final incubation, the ssDNA is collected and saved for sequencing.

Example 4: Binding On-Glass Counter Selection

The ssDNA molecules from the final round of positive on-glass SELEX (Example 2) are diluted to a concentration of 0.1 ng/μL in extraction buffer. The counter proteins of interest are dissociated in extraction buffer and diluted to 10000 ppm. 50 uL of the ssDNA solution is mixed with 50 μL of the counter target mixture and the mixture is incubated for 1 minute at room temperature with shaking. This ssDNA/protein mixture is then added to two wells of a fresh 16-well slide as previously described. Any ssDNA in the mixture that also has the capability to bind these undesired counter targets will bind the proteins and not bind the short complimentary sequences on the glass. After incubation for 1 minute at room temperature with shaking, the ssDNA/protein mixture is transferred to the next two wells of the same slide. This process is repeated for a total of eight incubations. Following the final incubation, the ssDNA is collected, cleaned as previously described, and saved for sequencing.

Example 5: Selection of Aptamers that Bind to Gluten

Gluten is found in wheat, buckwheat, barley, and rye, which is composed of two primary fractions, the water and alcohol soluble gliadins, and the insoluble glutenins (Journal of AOAC International 2013; 96, 1-8). Due to these solubility differences, aptamers that recognize the gliadin fraction are selected using the combined SELEX methods.

Gluten Extraction from Food

Several different extraction methods are used and compared (Fallahbaghery et al., J. Agric. Food Chem. 2017; 65, 2857-2866; and Ito et al., Anal Bioanal Chem. 2016; 408, 5973-5984). Surfactants, salts, and reducing agents are tested. The surfactants tested include 0.1% Tween 20 or 1% SDS. The salts tested include 25 mM NaCl and 5 mM MgCl₂ or 2 mM guanidinium HCl. The only reducing agent tested is 100 mM sodium sulfite, as other reducing agents present a serious health hazard for a consumer device.

In order to select the ideal extraction buffer, 24 different buffers were tested, and their extraction efficiency was measured by ELISA. The first round of testing was performed simply on wheat, while further rounds of testing used four different wheat-incurred foods: oatmeal, wine, ground pork, and ice cream. From this testing, the best gluten extraction buffer comprises 20 mM HEPES, 30% EtOH, 0.1% Tween20, 2 mM guanidinium HCl, 25 mM NaCl, and 5 mM MgCl₂.

Determining the Ratio of GO and ssDNA Molecules

The optimal ratio of GO to ssDNA molecules in the extraction buffer is determined to achieve the maximal recovery of ssDNA molecules during the selection process. The optimal ratio is 10-fold excess of GO to ssDNA, but the affinity of ssDNAs to GO varies depending on salt content of the buffer. A dilution curve of GO using the same amount of ssDNA revealed that a 2000:1 mass ratio of GO to ssDNA is needed in the gluten extraction buffer.

Every target protein has distinct cross-reactivity concerns. For gluten, several counter proteins classes are tested, including tree nuts, commonly used wheat replacements (arrowroot, rice flour, buckwheat), and the other major allergens (egg, milk, soy).

Example 6: Selection of Sequences as Peanut Control

Control sequences can be used in a detection assay, for example in an allergen detection assay to measure the total protein. Signals from control sequences may be incorporated into the assay algorithm in place of, or in addition to the fiducials. In this example, control sequences for peanut detection (i.e. peanut control sequences) were selected from the ssDNA library using SELEX methods as described herein. The criteria for control sequences include: 1) having similar response to a corresponding matrix, e.g., food type, as the target such as AraH1 (peanut allergen); 2) having no or litter response to the target material, e.g., peanut; 3) having no binding to either the aptamer against the target (AraH1) or its anchor sequences.

To select peanut control sequences, repeated selections with different binding materials were performed. Before each round of selection, a counter selection against 10,000 ppm peanut was performed and the collected sequences from each round of the counter selection were used. Table 16 lists the repeated selections with different binding materials.

TABLE 16 Materials and selections for peanut control sequences Round Selection Materials SELEX step 1 1000 ppm Actin GO-SELEX 2 1000 ppm BSA GO-SELEX 3 1000 ppm Soy flour GO-SELEX 4 1000 ppm Tannin GO-SELEX 5-12 Repeat rounds 1-4 twice 13 100 ppm Actin Glass-SELEX 14 100 ppm BSA Glass-SELEX 15 100 ppm Soy flour Glass-SELEX 16 100 ppm Tannin Glass-SELEX 17 100 ppm Peanut Glass-SELEX 18 No protein Glass-SELEX

Sequences collected from Rounds 16, 17 and 18 were sequenced and tested. The heat maps, predicted binding to an aptamer specific to the peanut allergen protein AraH1 (AraH1 probe) and the anchor sequences, and folded structures of each sequence were analyzed. Table 11 lists the top 1000 hits from the selection. 13 control sequences (Table 17) were picked and further characterized,

TABLE 17 Control aptamers for peanut control materials Control SEQ sequence Sequence (5′-3′) ID NO PC14 TAGGGAAGAGAAGGACATATGATGTCGTGACTG 39016 GCTAGCTGGACATGCACTGCTTGACTAGTACAT GACCACTTGA PC36 TAGGGAAGAGAAGGACATATGATGCACTGGCTG 39038 ACCTACACGTGGACGATGTGTTGACTAGTACAT GACCACTTGA PC41 TAGGGAAGAGAAGGACATATGATGCACGCCGAT 39043 GCCCTCATGTGGCCGTGGATTGACTAGTACATG ACCACTTGA PC48 TAGGGAAGAGAAGGACATATGATGACGACACGA 39050 CCTTCAAGCATGGCCTAGCGTTGACTAGTACAT GACCACTTGA PC54 TAGGGAAGAGAAGGACATATGATGGACGCAACG 39056 TACCGTATCGTGGCCATGTGTTGACTAGTACAT GACCACTTGA PC55 TAGGGAAGAGAAGGACATATGATGCACGTACGC 39057 CTTGCCTATCTGTGCTCATGTTGACTAGTACAT GACCACTTGA PC58 TAGGGAAGAGAAGGACATATGATGGCATGCGCT 39060 GGGTAGTGATCACGTACGGTTTGACTAGTACAT GACCACTTGA PC60 TAGGGAAGAGAAGGACATATGATCGTACCGCAA 39062 GTGACGTGTCCGTGCCGTGATTGACTAGTACAT GACCACTTGA PC66 TAGGGAAGAGAAGGACATATGATGTCATGCGCG 39068 TACCATCGAGGGGGCGTGGATTGACTAGTACAT GACCACTTGA PC77 TAGGGAAGAGAAGGACATATGATGGACTGAACG 39079 TACTGCCAGGTGAGCATGCATTGACTAGTACAT GACCACTTGA PC85 TAGGGAAGAGAAGGACATATGATCGATGGTACG 39087 AACGCCACGTCATGCGGTCATTGACTAGTACAT GACCACTTGA PC87 TAGGGAAGAGAAGGACATATGATGCGTGTCAGC 39089 AATACGTCCTCATCTGCCCGTTGACTAGTACAT GACCACTTGA PC96 TAGGGAAGAGAAGGACATATGATGGACAACGTG 39098 GCTGGTAGGTATCGTGGGCATTGACTAGTACAT GACCACTTGA

None of these peanut control sequences bind to the aptamer specific to AraH1 (AraH1 probe) up to the concentration of 100 nM.

The binding of the control sequences to an anchor sequence (AAAAATCAAGTGGTC; SEQ ID NO. 52003), the interference of each sequence with the binding of the AraH1 probe to peanut, and the affinity of each sequence to peanut, were evaluated. The data showed that three sequences PC36, PC60 and PC87 have minimal response to 5000 ppm peanut when tested at a concentration of 100 nM.

Two food types including sugar free wafer and strawberry poptart were compared for response to AraH1 aptamer and peanut control sequences PC36, PC60 and PC87 (each at a concentration of 100 nM). The foods were spiked with either 0 ppm or 5000 ppm peanut. The data indicate the AraH1 aptamer creates high signal for wafer but a lower signal for poptart.

TABLE 18 signals of AraH1 aptamer and peanut control sequences poptart wafer Ration 0 5000 % of T- 0 5000 % of T- (Wafer:poptart) sequence ppm ppm change test ppm ppm change test 0 ppm AraH1 1.9 1.8  93% 0.032 6.8 1.7 26% 0.003 3.5 aptamer PC36 5.9 6.5 109% 0.612 23.3 15.8 68% 0.069 3.9 PC60 5.4 5.3 100% 0.981 9.5 7.6 80% 0.981 1.8 PC87 103.9 102.3  99% 0.920 127.6 132.5 104%  0.791 1.2 

1. A method for identifying an aptamer that specifically binds to a target of interest comprising: (a) preparing an input DNA library comprising a plurality of single stranded DNA (ssDNA) molecules, each of which comprises a central randomized nucleic acid sequence flanked by a constant sequence at the 5′ end and a constant sequence at the 3′ end, the constant 5′ end and the constant 3′ end functioning as primers; (b) selecting, from the input DNA library, a first pool of ssDNA molecules that substantially bind to the target; (c) selecting a second pool of ssDNA molecules, from the first target binding pool of ssDNA molecules, that do not substantially hybridize to their complementary sequences in the presence of the target; (d) counter-selecting a third pool of ssDNA molecules, from the second positive binding pool of ssDNA molecules obtained, that do not hybridize to the complementary sequences in the absence of the target, and a fourth pool of sequences that substantially bind to counter targets; and (e) subtracting the ssDNA molecules in the third and fourth pools from the second positive binding pool of ssDNA molecules, and identifying ssDNA molecules that specifically bind to the target of interest.
 2. The method of claim 1, wherein the first pool of ssDNA molecules that substantially bind to the target material is selected through the steps of: (i) contacting the input ssDNA library with a target material wherein complexes are formed between the target and a plurality of ssDNA molecules present in the input library; (ii) partitioning the ssDNA:target complexes formed in step (i) from unbound ssDNA molecules using a Graphene Oxide (GO) solution, and isolating the ssDNA molecules in the complexes to produce a subset of ssDNA molecules for the target; (iii) contacting the subset of ssDNA molecules in (ii) with the same target wherein complexes are formed between the target and a second plurality of ssDNA molecules present in the subset of ssDNA molecules to generate a second subset group of ssDNA molecules for the target; and (iv) optionally repeating steps (ii) to (iii), one, two, three, four or more rounds to produce a respective third, fourth, fifth, sixth or more subset group of ssDNA molecules, thereby producing the enriched pool of ssDNA molecules that substantially bind to the target after the final round.
 3. The method of claim 2, wherein the second positive binding pool of ssDNA molecules is selected through an on-chip positive selection process using the first target binding pool of ssDNA molecules, the same target and a solid support that is coated with short oligonucleotides that are complementary to the constant sequence of the ssDNA molecules, the on-chip positive selection process comprising the steps of: (i) mixing the first target binding pool of ssDNA molecules with the same target in a buffer solution and inducing them to bind to each other; (ii) contacting the mixture of step (i) with the solid support of which the surface is covalently coated with short oligonucleotides that are complementary to the constant sequence of the ssDNA molecules; (iii) collecting a flow-through containing ssDNA:target complexes that are not bound to the solid support; (iv) optionally contacting the collected flow-through again with the solid support coated with the complementary oligonucleotides for two, three, four, five, six, seven, eight, or more times; and (v) removing the target and collecting an enriched subset of ssDNA molecules after the final incubation.
 4. (canceled)
 5. The method of claim 3, wherein the third pool of ssDNA molecules is selected by an on-chip non-binding counter process using the second positive binding pool of ssDNA molecules and a solid support that is coated with short oligonucleotides that are complementary to the constant sequence of the ssDNA molecules.
 6. The method of claim 3, wherein the fourth pool of ssDNA molecules is selected through an on-chip counter binding process using the second positive binding pool of ssDNA molecules, one or more counter targets and a solid support that is coated with short oligonucleotides that are complementary to the constant sequence of the ssDNA molecules.
 7. The method of claim 1, wherein the method further comprises: (f) amplifying and sequencing the ssDNA molecules in each pool obtained in steps (b) to (d); and (g) generating a sequence map for each pool of ssDNA molecules and analyzing the sequence information and selecting a final pool of ssDNA molecules that specifically and preferentially bind to the target of interest in the presence of the complementary sequences.
 8. The method of claim 7, wherein the step (g) comprises: (i) amplifying all the ssDNA molecules in the first, second, third and fourth pools, and barcoding each sequence from each pool; (ii) pooling together the sequences from each pool and running sequencing together; (iii) analyzing the data from (ii) and separating data for each sequence into the original pool according to the barcode information; (iv) generating heat maps for each individual pool that represent the frequency of each sequence in the pool; and (v) subtracting the sequences in the heat maps of the third pool of ssDNA molecules and the sequences in the fourth pool of ssDNA molecules, from the heat maps of the second positive binding pool of ssDNA molecules, wherein the final pool of the sequences after step (v) represents aptamer candidates that specifically bind to the target of interest and preferentially bind to the target of the interest in competing the binding of various short complementary sequences.
 9. The method of claim 8, wherein the method further comprises subtracting any sequences from an artifact pool of ssDNA molecules from the first target binding pool of ssDNA molecules, wherein the artifact pool of ssDNA molecules is generated by PCR amplification and strand separation of the input DNA library, representing the sequences that are over-amplified by PCR amplification.
 10. The method of claim 9, wherein the sequences of the final pool are analyzed for sequence similarities and secondary structures.
 11. The method of claim 7, wherein the sequences of ssDNA molecules in each pool are amplified by PCR using a pair of Cy5 labeled primers and biotinylated primers.
 12. The method of claim 1, wherein the target is an allergen.
 13. An aptamer that binds to an allergen with high specificity and affinity, wherein the aptamer does not hybridize to its complementary sequences in the presence of the target allergen.
 14. (canceled)
 15. The aptamer of claim 13, wherein the allergen is peanut and the aptamer specific to peanut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs.3 to
 4002. 16. (canceled)
 17. The aptamer of claim 13, wherein the allergen is almond and the aptamer specific to almond comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 4003 to
 8002. 18. (canceled)
 19. The aptamer of claim 13, wherein the allergen is brazil nut and the aptamer specific to brazil nut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 8003 to
 12002. 20. (canceled)
 21. The aptamer of claim 13, wherein the allergen is cashew and the aptamer specific to cashew comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 12003 to
 16002. 22. (canceled)
 23. The aptamer of claim 13, wherein the allergen is hazelnut and the aptamer specific to hazelnut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 16003 to
 20002. 24. (canceled)
 25. The aptamer of claim 13, wherein the allergen is pecan and the aptamer specific to pecan comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 20003 to
 24002. 26. (canceled)
 27. The aptamer of claim 13, wherein the allergen is pistachio and the aptamer specific to pistachio comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 24003 to
 28002. 28. (canceled)
 29. The aptamer of claim 13, wherein the allergen is walnut and the aptamer specific to walnut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28003 to
 32002. 30. (canceled)
 31. The aptamer of claim 13, wherein the allergen is a mix of nuts and the aptamer specific to the mixed nuts comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 32003 to
 36002. 32. (canceled)
 33. The aptamer of claim 31, wherein the mixed nuts comprise peanut, almond, brazil nut, cashew, hazelnut, pistachio, pecan and walnut.
 34. The aptamer of claim 13, wherein the allergen is gluten and the aptamer specific to gluten comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 40003 to
 44002. 35. (canceled)
 36. The aptamer of claim 13, wherein the allergen is whey and the aptamer specific to whey comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 44003 to
 48002. 37. (canceled)
 38. The aptamer of claim 13, wherein the allergen is casein and the aptamer specific to casein comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 48003 to
 52002. 39. (canceled)
 40. (canceled)
 41. A control aptamer that recognizes a panel of control materials that are used for peanut allergen comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 36003 to
 40002. 42. (canceled)
 43. A signaling polynucleotide (SPN) comprising an aptamer sequence that binds to an allergen with high specificity and affinity, wherein the aptamer sequence does not hybridize to its complementary sequences in the presence of the target allergen, and a fluorophore conjugated to one end of the aptamer sequence, and a short oligonucleotide sequence that is complementary to the aptamer sequence or a portion of the aptamer sequence.
 44. The SPN of claim 43, wherein the fluorophore is Cy5, Texas red or Alexa Fluor
 647. 45. (canceled)
 46. The SPN of claim 44, wherein the allergen is peanut and the aptamer specific to peanut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 3 to 4002; wherein the allergen is almond and the aptamer specific to almond comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 4003 to 8002; wherein the allergen is brazil nut and the aptamer specific to brazil nut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 8003 to 12002; wherein the allergen is cashew and the aptamer specific to cashew comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 12003 to 16002; wherein the allergen is hazelnut and the aptamer specific to hazelnut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 16003 to 20002; wherein the allergen is pecan and the aptamer specific to pecan comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 20003 to 24002; wherein the allergen is pistachio and the aptamer specific to pistachio comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 24003 to 28002; wherein the allergen is walnut and the aptamer specific to walnut comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 28003 to 32002; wherein the allergen is a mix of nuts and the aptamer against the nuts comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 32003 to 36002; wherein the allergen is gluten and the aptamer specific to gluten comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 40003 to 44002; wherein the allergen is whey and the aptamer specific to whey comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 44003 to 48002; or wherein the allergen is casein and the aptamer specific to casein comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 48003 to
 52002. 47. (canceled)
 48. (canceled)
 49. (canceled)
 50. (canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled)
 55. The SPN of claim 46, wherein the mixed nuts comprise peanut, almond, brazil nut, cashew, hazelnut, pistachio, pecan and walnut.
 56. (canceled)
 57. (canceled)
 58. (canceled)
 59. (canceled)
 60. The SPN of claim 43, wherein the short complementary sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 52003 to
 52042. 61. A detection sensor comprising: (i) signaling polynucleotide (SPN), wherein the SPN comprises an aptamer sequence that binds to an allergen with high specificity and affinity and that does not hybridize to its complementary sequences in the presence of the target allergen, and (ii) a short nucleic acid sequence that is printed on a solid surface, wherein the short nucleic acid sequence is complementary to the SPN.
 62. (canceled)
 63. (canceled)
 64. The detection sensor of claim 61, wherein the allergen is peanut, almond, brazil nut, cashew, hazelnut, pecan, pistachio, gluten, whey or casein.
 65. The detection sensor of claim 61 further comprising a control nucleic acid sequence, wherein the control sequence has the features including: i) no binding affinity to the target of interest; ii) no binding affinity to the target specific aptamer and iii) no binding affinity to the short anchor sequences on the solid surface.
 66. A detection kit comprising, (a) a signaling polynucleotide (SPN) comprising an aptamer sequence that binds to a target of interest with high specificity and affinity and that does not hybridize to its complementary sequences in the presence of the target of interest; (b) a solid support of which the surface is coated with short nucleic acid sequences that are complementary to the sequence of the aptamer; and (c) one or more buffer solutions.
 67. (canceled)
 68. The detection kit of claim 66 further comprising a SPN comprising an aptamer sequence that binds to a control material.
 69. A method for detecting the presence, and/or absence of an allergen in a food sample comprising: (a) preparing a food sample solution wherein the solution comprising a SPN comprising an aptamer that specifically binds to said allergen an allergen and that is labeled with a fluorophore; (b) contacting the mixture of the sample and SPN to a solid support that is coated with short nucleic acid sequences that are complementary to the aptamer sequence; and (c) measuring fluorescence signals and detecting the presence and/or absence of the allergen of interest in the food sample.
 70. The method of claim 69 further comprising a step of (d) measuring the total protein from the food sample using a SPN comprising an aptamer that bind to the allergen control material.
 71. The method of claim 69, wherein the allergen is peanut.
 72. The method of claim 71, wherein the SPN that specifically binds to peanut comprising a nucleic acid sequence selected the group consisting of SEQ ID NOs. 3 to
 4002. 73. The method of claim 72, wherein the SPN that binds to peanut control material comprising a nucleic acid sequence selected the group consisting of SEQ ID NOs. 36003 to
 40002. 